Analytical Methods

Acetylcholinesterase based biosensor for monitoring of Malathion andAcephate in food samples: A voltammetric study

P. Raghu a, T. Madhusudana Reddy a,⇑, K. Reddaiah a, B.E. Kumara Swamy b, M. Sreedhar c

a Electrochemical Research Laboratory, Department of Chemistry, S.V.U. College of Sciences, Sri Venkateswara University, Tirupati 517502 Andhra Pradesh, Indiab Department of P.G. Studies and Research in Industrial Chemistry, Kuvempu University, Shankaraghatta, 577451 Shimoga, Karnataka, Indiac Pesticide Residue Analysis Laboratory, Regional Plant Quarantine Station, Meenambakkam, Chennai 600027, Tamilnadu, India

a r t i c l e i n f o

Article history:Received 2 March 2012Received in revised form 8 July 2013Accepted 10 July 2013Available online 17 July 2013

Keywords:BiosensorAcetylthiocholine chlorideCarbon paste electrodeImmobilisationMalathion and Acephate

a b s t r a c t

Acetylcholinesterase (AChE) biosensor was developed through silica sol–gel (SiSG) immobilisation ofAChE on the carbon paste electrode (CPE) and used as working electrode. AChE catalyses the cleavageof acetylthiocholine chloride (ASChCl or substrate) to thiocholine, which was oxidised to give a disul-phide compound by dimerisation at 0.60 V versus saturated calomel electrode. All the experiments werecarried out in 0.1 M phosphate buffer solution (PBS) at pH 7.0 and 0.1 M KCl solution at room tempera-ture. The limit of detection and limit of quantification values were found to be 0.058 ppm, 0.044 ppm and0.194 ppm, 0.147 ppm for Malathion and Acephate, respectively. The response of the biosensor showed agood linearity range with an incubation time of 4 min for Malathion and Acephate, respectively. This bio-sensor was used for the direct determination of pesticides without any pretreatment and it requires lesstime for analysis.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Organophosphorous insecticides are very toxic compounds,intensively used in agriculture. Their existence in the environmentis very harmful to human health as they irreversibly inhibit thecatalytic active sites of AChE, an essential enzyme that permitsthe transmission of electric signals in the nervous system of mostanimal beings. However, there is a fear towards the poisoning ofdrinking water sources and food samples by neurotoxic agents,which can produce acute poisoning even at very low concentra-tions (Hildebrandt et al., 2008a). In recent years, growing attentionhas been to the development of reliable, fast and inexpensive ana-lytical systems to monitor pesticides from environmental samples.

Malathion (ML) and Acephate (AP) are organophosphorousinsecticides. These pesticides are widely used in agriculture, resi-dential landscaping, public relation areas and in public health pestcontrol programs such as mosquito eradication. The misuse oroverdose of these pesticides results in contamination of fields,crops, water and air. Both the compounds are relatively insolublein water, poorly soluble in petroleum ether and mineral oils, andreadily soluble in most organic solvents. The physical, chemicaland biological factors are influencing the distribution of above pes-ticides in air, water, soil and organisms in the environment. Thesepesticides are also readily taken into the body through skin,

inhalation, even though the amount absorbed will depend onwhere the exposure occurs on the body. Organophosphates suchas ML and AP are inhibitors of cholinesterases.

Many methods have been developed in the last few years for thedetermination of pesticides. Most of these are based on a separationby gas (Maria Dolores, Ana, Amadeo, Luis, & Mariano, 2001) or li-quid chromatography (Wang, Wang, & Yuan, 1999), spectropho-tometry (Rodrignez, Monferrer-Pons, Esteve Romero, GarciaAlvarez, & Ramis Rames, 1997; Tuxelli, Bag, & Turker, 2001), infra-red spectroscopy (Capitan-Valley, Deheidel, & Avivad, 1998; Dagh-bouche, Garrigues, & Guardia, 1995; Mc Garvey, 1993; MurilloPulgarin, Molina, & Fernandez Lopez, 2006), fluorescence spectrom-etry (De kok & Hiemstra, 1992), mass spectrometry (Newsome, Lau,Ducharme, & Lewis, 1995), high performance liquid chromatogra-phy (HPLC), UV or electrochemical detectors. Many of the abovementioned methods are accurate and selective, but they require rel-atively expensive instrumentation and high consumption of toxicorganic reagents, becoming expensive and time consuming. Thedevelopment of alternative methods, which minimises the con-sumption of reagents and decreases in the time of analysis withthe quality of measurements, is of great interest. A numerous bio-sensing methods have been developed for the detection of pesti-cides by using enzyme based and affinity – based sensors as wellas several types of transducers (Suwansa-ard et al., 2005). Amongthe electrochemical sensors, the enzyme modified electrode playsa vital role in the development of highly sensitive, selective, lowcost and chemical analysis with short time biosensor.

0308-8146/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.foodchem.2013.07.047

⇑ Corresponding author. Tel.: +91 877 2289303.E-mail address: tmsreddysvu@gmail.com (T. Madhusudana Reddy).

Food Chemistry 142 (2014) 188–196

Contents lists available at ScienceDirect

Food Chemistry

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

Enzyme sensors based on the inhibition of AChE with potentio-metric or amperometric detection (Kindervater, Kunnecke, & Sch-mid, 1990; Leon-Gonzales & Townshend, 1991; Martorell,Cespedes, Martinez-Fabregas, & Alegret, 1994; Rouillon, Mionelto,& Marty, 1992; Skaladal, 1992) have been developed for the deter-mination of organophosphorous and carbamate pesticides. Theimmobilisation of AChE in biosensor technology involves differenttypes of methods. Such as sol–gel entrapment, covalent binding,adsorption and encapsulation of sensing agents with in a polymers.Among these sol–gel immobilisation can be preferred since theother immobilisation methods are tedious, result in poor stability,and require expensive reagents, enzyme leaking and loss of en-zyme activity. So many sol–gel derived enzyme biosensors havebeen developed to monitor glucose, lactate, cholesterol, dopamine,H2O2, phenols and urea (Pandey, Upadhyay, Tiwari, Singh, & Tri-pathi, 2001; Park, Lwuoha, Smyth, Freoney, & Mc Shane, 1997;Sampath & Lev, 1996; Yao & Takashima, 1998; Park, 1999; Li, Chia,Goh, & Tan, 1998; Pandey & Singh, 2001).

In this work, we demonstrated that acetylthiocholine chloride(ASChCl) used as a substrate for the enzyme catalysed reaction.We developed a biosensor by using TEOS sol–gel system dopedwith AChE towards the determination of ML and AP pesticides inspiked food samples. The parameters such as operational, linearrange, pH, response and response time have been investigated.

2. Experimental

2.1. Apparatus

The electrochemical measurements were conducted in a threeelectrodes cell at the room temperature 25 ± 2 �C. The workingelectrode was the enzyme immobilised carbon paste electrode(AChE–SiSG–CPE). The reference electrode was the saturated calo-mel electrode system and platinum wire was used as the auxiliaryelectrode. Measurements were carried out using CH – Electro-chemical Analyzer (Model CHI – 660D, CH Instruments, USA).

2.2. Materials

All chemicals were obtained from commercial sources and usedwithout further purification. Acetyl cholinesterase (E.C. 3.1.1.7type-VI-S/1.5 mg, electric eel source, 500 U/1.5 mg) and acetylthi-ocholine chloride were purchased from Sigma–Aldrich chemicalsCo. Missouri, USA. Malathion and Acephate were obtained fromRiedel–deHaen, Fluka, Missouri, USA. The pesticide stock solutionwas prepared in acetone (GR grade solution, obtained from Merckspecialities Pvt. Ltd., Mumbai (INDIA). Tetraethyl orthosilicate(TEOS), cetyltrimethyl ammonium bromide (CTAB), Triton-X-100were obtained from Sigma–Aldrich chemicals Co. Missouri, USA.The graphite fine powder was procured from Loba Chemie Pvt.Ltd., Mumbai (INDIA) and silicon oil from HiMedia LaboratoriesPvt. Ltd., Mumbai (INDIA). Phosphate buffer solution (PBS) wasprepared by mixing 0.1 M sodium dihydrogen phosphate monohy-drate and 0.1 M disodium hydrogen phosphate. All the aqueous

solutions were prepared with double distilled water. The enzymestock solution and working solutions of chemicals were stored incool place.

2.3. Preparation of bare carbon paste electrode

The bare carbon paste electrode was prepared by hand mixingof 70% graphite powder and 30% silicon oil in an agate mortar toproduce a homogenous carbon paste. The paste was packed intothe cavity of homemade PVC (3 mm in diameter) and thensmoothed on a weighing paper. The electrical contact was providedby copper wire connected to the paste at the end of the tube (Chan-dra, Kumara Swamy, Gilbert, & Sherigara, 2010; Chithravathi,Kumara Swamy, Cahndra, Mamatha, & Sherigara, 2010; Gilbert,Kumara Swamy, Chandra, & Sherigara, 2009).

2.4. Biosensor/AChE–SiSG–CPE preparation

A homogenous TEOS silica sol was prepared by mixing 2 ml ofTEOS, 1 ml of H2O, 50 lL of 0.1 M HCl, and 25 lL of 10% Triton-X-100. The mixture was stirred for 1 h and homogenised beforeeach usage and stored in cool place.

The 5 lL of stock sol–gel solution was mixed with 45 lL ofphosphate buffer containing 0.5 U of enzyme stock solution andfrom this 5 lL of enzyme sol of concentration 0.05 U was spreadon the electrode surface. This electrode was allowed to keep forabout 3–5 min at room temperature. Finally the electrodewas cleaned with PBS (pH 7.0) and was used for further experi-mental procedures (Anitha, Venkata Mohan, & Jayarama Reddy,2004).

2.5. Sample collection

The food samples listed in Table 1B were chosen for the deter-mination of pesticide residues. The food samples were taken fromthe fields near Swarnamuki River (Chittoor (District), Andhra Pra-desh, India). The samples were cleaned with distilled water,chopped and removed outer membrane. From this five grams ofeach chopped or grained samples were added to 10 ml PBS (pH7.0) and stirred for 1 h at room temperature. This sample solutionwas filtered and the filtrate was dissolved in acetone. These sam-ples were spiked with a known concentration of ML and AP derivedfrom stock solution and used immediately for analysis.

3. Results and discussion

3.1. Cyclic voltammetric behaviour of biosensor

The fabricated AChE based biosensor entrapped through sol–gelimmobilisation method onto the carbon paste electrode (CPE) wasshown through the mechanism in Fig. 1. The electrochemicalbehaviour of biosensor was evaluated by using acetylthiocholinechloride (ASChCl) as a substrate. The enzyme catalysed reactionand anionic oxidation of thiocholine was shown below.

P. Raghu et al. / Food Chemistry 142 (2014) 188–196 189

All sensor experiments were carried out in 0.1 M PBS/pH (7.0),0.1 M KCl at room temperature by employing cyclic voltammetry(CV). Fig. 2A shows the cyclic voltammograms of the biosensor inthe absence and presence of the substrate (1–6 mM) in PBS (pH7.0) at the scan rate of 5 mVS�1. In absence of substrate no peakcan be observed even up to 1.0 V with biosensor. When the sub-strate is present a larger anodic peak was observed at 0.60 V thiswas due to the oxidation of thiocholine, which inturn producesdimmer structure (Andreescu, Barthelmebs, & Marty, 2002) andwas shown in (R2). The biosensor response increases with the sub-strate concentration until a saturation point is reached and wasseen through the plateau in inset of Fig. 2A. The kinetics of theimmobilised enzyme showing Michaelis–Menten plot characteris-tics, was diphasic in nature. We observed linear dependency of ratefor smaller substrate concentration (first order) by a correlationcoefficient very close to unity ([S] << Kapp

m , where [S] is the substrateconcentration & Kapp

m is the apparent Michaelis–Menten constant).For higher substrate concentration, the rate was independent ofconcentration (zero order, [S] >> Kapp

m ). The apparent Michaelis–Menten constant (Kapp

m ) was 0.57 mM which was obtained fromthe linear portion of the calibration plot using Lineweaver–Burkequation.

In order to support the enzyme catalysed hydrolysis reaction,the variation of the peak potentials as a function of the scan ratewas analysed. The anodic peak potentials depend linearly on thelogarithm of the scan rate as predicted by Eqs. (1) & (2) proposedby (Aoki, Akimoto, Tokuda, Matsuda, & Osteryoung, 1984).

Epa ¼ Eo þm½0:78þ lnðD1=2=KoÞ � 0:5 ln m� þ 0:5m ln t ð1Þ

m ¼ RT=½ð1� aÞnF� ð2Þ

Where ‘Eo’ is the formal potential of acetylthiocholine, ‘D’ is thediffusion coefficient, ‘Ko’ is the heterogeneous standard rate con-stant, ‘a’ is the energy transfer coefficient and ‘n’ is the numberof electrons transferred during the heterogeneous reaction. R, Tand F are the universal gas constant, absolute temperature and Far-aday constant respectively. The formal potential of ASChCl was de-duced from the intercept of Ep Vs t plot. By using the value ofacetylthiochline diffusion coefficient 0.12 � 10�6 cm2 S�1 and fromthe intercept of Epa Vs ln t plot, the value of Ko was calculated as0.00026 cm S�1. From Eq. (3) (Bard & Faulkner, 2000), it is possibleto calculate the value of the energy transfer coefficient (a) as 0.35.The ‘a’ value and the slope obtained from the plot of Epa Vs ln t wassubstituted in Eq. (2). From the calculations, the number of elec-trons involved during ASChCl oxidation process was found to be1.94 (approx. 2). From this result, we conclude that ASChCl oxida-tion occurs through, two electron transfer process as indicated in(R2).

ip ¼ 0:227 FAC�oKo exp½�af ðEp � EoÞ� ð3Þ

Fig. 1. Immobilization of AChE through sol–gel process on the CPE.

Fig. 2. (A) Cyclic voltammograms of AChE–SiSG–CPE in 0.1 M PBS (pH 7.0)/0.1 MKCl and at the scan rate of 5 mV s�1 (a) without substrate (b) 1.0 (c) 1.5 (d) 2.0 (e)2.5 (f) 3.0 (g) 4.0 (h) 5.0 and (i) 6.0 mM substrate and the calibration plot has beenshown in the inset. (B) Effect of pH on the anodic peak potential (Epa) and insetshows plot of Ipa Vs pH to 1 mM substrate. (C) The variation of biosensor responseagainst the concentration of enzyme on AChE–SiSG–CPE.

190 P. Raghu et al. / Food Chemistry 142 (2014) 188–196

Fig. 3. (A) Inhibition of AChE in the presence of pesticides. (B) Differential-pulse-voltammograms recorded with AChE–SiSG–CPE in a 1 mM ASChCl + 0.1 mM PBS (pH 7.0)/0.1 MKCl after addition of ML in different concentrations (a) 0 (b) 0.07 ppm (c) 0.15 ppm (d) 0.4 ppm (e) 1.0 ppm (f) 1.3 ppm. Inset: calibration plot of % inhibition against MLconcentration with an incubation time of 4 min. (C) Differential-pulse-voltammograms recorded with AChE–SiSG–CPE in a 1 mM ASChCl + 0.1 mM PBS (pH 7.0)/0.1 M KCl afteraddition of AP in different concentrations (a) 0 (b) 0.1 ppm (c) 0.2 ppm (d) 0.35 ppm (e) 0.5 ppm (f) 0.75 ppm (g) 0.85 ppm. Inset: analytical curve of % inhibition against APconcentration with an incubation time of 4 min. (D) and (E) Effect of incubation time on various inhibitor concentrations in 0.1 M PBS (pH 7.0)/0.1 M KCl for ML and AP, respectively.

P. Raghu et al. / Food Chemistry 142 (2014) 188–196 191

Where A is the electrode surface area, C�o is the ASChCl concentra-tion and f = F/RT.

3.2. Effect of pH

The pH of the solutions containing substrates can affect the en-zyme activity. The denaturation of enzyme can occur at extremepH values due to conformational changes observed in a native ter-tiary structure of AChE enzyme. The response of the AChE – biosen-sor as a function of pH has been studied between pH 5.5–8.5 andwas shown in inset of Fig. 2B. The enzyme activity decreasesapproximately 70% at pH 5.5 compared to that of pH 7.0. Whenthe pH is more than 8.0 the response is very small. The maximumcurrents appeared at pH 7.0. The anodic peak potentials shifted toless positive side with increasing the pH of the buffer solution andwere shown in Fig. 2B. The graph has good linearity with slope of53 mV/pH. This slope value was close to the theoretical slope of(59 mV/pH), which is in accordance with Nernst equation fortransfer of equal number of electrons and protons in the reaction(Chandra, Kumara Swamy, Gilbert, & Sherigara, 2010; Gilbert,Kumara Swamy, Chandra, & Sherigara, 2009).

3.3. Effect of enzyme concentration

Fig. 2C shows the response of the biosensor and was found to bedependent on the amount of immobilised enzyme on the surface ofCPE. All experiments were carried out in 1 mM substrate solutionprepared in 0.1 M PBS (pH 7.0). It can be concluded from the ob-tained data that the enzyme concentration from 0.03 to 0.3 U intothe sol–gel layer, produced increased response of the biosensor.The enzyme concentration of 0.05 U was chosen as best compro-mise between a low enzyme loading and enough substrate signals.In fabrication of biosensor the lowest feasible concentration of en-zyme was necessary towards the determination of pesticides withlower detection limits (Mohammadi, Amine, Cosnier, & Mousty,2005; Shane, Mousty, & Cosnie, 2004).

3.4. Determination of pesticides using AChE–SiSG–CPE/biosensor

AChE biosensor could potentially provide important informa-tion about the relative inhibitory effect of ML and AP. In order to

obtain lower detection limit and for rapid analysis, incubation timeof 4 min was selected for inhibition measurements of both pesti-cides. Two pesticides are investigated under the optimum condi-tions (0.1 M PBS, 0.1 M KCl, pH 7.0 and 1 mM substrate). Thestandard solutions of the two pesticides were freshly prepared be-fore the experiments to avoid decomposition.

Measurement of organophosphorus pesticides was carried outin a two step procedure. The first step involves in measuring theresponse of biosensor, when the substrate was added to the bufferand this value is corresponds to Ii (current before the inhibition ofpesticide). In the second step the electrode was washed with thesame buffer and was placed in the sample containing a known con-centration of pesticide solution and measured the response, thissecond value corresponds to IF (current after the inhibition of pes-ticide). The percentage of enzyme inhibition and residual enzymeactivity percentage was determined according to the following for-mula (Anitha et al., 2004).

Inhibition %ðI %Þ ¼ ½ðIi � IFÞ=Ii� � 100 ð4Þ

Residual enzyme activity %ðREA %Þ ¼ ½IF=Ii� � 100 ð5Þ

The electrochemical determination of organophosphorus pesti-cides was performed through the inhibition reaction of AChE. Afterinhibition, the thiocholine produced was in less concentration fromthe enzymatic reaction and decrease of oxidation peak current wasobserved and this was due to the blocking active sites of the AChE.The AChE has active site gorge that penetrates basically halfwaythrough the enzyme and it consists of two sub sites, namely an an-ionic site and an esteratic site. The anionic site is located at theopening of the gorge with Tryptophan-84 (Trp-84) and Phenylala-nine-330 (Phe-330). The esteratic site is formed by Serine-220(Ser-200), Histidine-440 (His-440) and Glutamate-327 (Glu-327)(i.e. catalytic triad) and was blocked by the pesticides throughthe proposed mechanism. In the first step the proton of the serineis accepted by imidazolic nitrogen of the hystidine residue (His440). The proton of the imidazol ring is exchanged with anion ofglutamate (Glu 327). The activated serine is a strong nucleophile,which can attack the carbonyl/thiocarbonyl of the pesticide. Thenegative charge formed on carbonyl oxygen/sulphur of pesticidesis rearranged to produce blocked serine residue and –R1H. Themechanism of inhibition of pesticide was illustrated in the Fig. 3A.

Table 1AThe % inhibition of AChE for unspiked and spiked food samples.

Samplematrix

na Unspiked meaninhibition%

Pesticide Spiked(ppm)

Spiked meaninhibition %

(Spiked–unspikedvalue) %

Expected(ppm)

Found(ppm)

Recovery%

Grape 3 23 AP 0.2 54 31 0.25 0.22 88 ± 1.43 27 ML 0.6 62 35 0.65 0.61 94 ± 1

Apple 3 19 AP 0.2 52 33 0.24 0.21 87 ± 1.789 ± 3b

3 24 ML 0.6 61 37 0.63 0.6 95 ± 199 ± 3b

Mango 3 18 AP 0.2 50 32 0.22 0.18 82 ± 23 22 ML 0.6 59 37 0.61 0.57 93 ± 1

Orange 3 41 AP 0.2 53.5 12.5 0.28 0.21 75 ± 2.53 48 ML 0.6 67 19 0.87 0.71 82 ± 2

Banana 3 17 AP 0.2 50.5 33.5 0.24 0.18 75 ± 33 21.5 ML 0.6 75 53.5 0.67 0.59 88 ± 2

Tomato 3 17.5 AP 0.2 52 34.5 0.25 0.2 80 ± 23 18 ML 0.6 63.5 45.5 0.65 0.62 95 ± 2

Rice 3 19 AP 0.2 54 35 0.25 0.23 92 ± 23 22 ML 0.6 61 39 0.68 0.60 88 ± 4.5

Wheat 3 24 AP 0.2 51 27 0.26 0.21 81 ± 4.53 28 ML 0.6 63 35 0.73 0.62 85 ± 3

a n = number of assays.b Results obtained with molecularly imprinted matrix solid phase dispersion–gas chromatography (MIMSPD–GC) (Wang et al., 2013) was compared with the present

method.

192 P. Raghu et al. / Food Chemistry 142 (2014) 188–196

Fig. 4. Bar diagram showing theoretical and experimental inhibition values of (A) ML – 0.6 ppm and (B) AP – 0.2 ppm in different food samples.

P. Raghu et al. / Food Chemistry 142 (2014) 188–196 193

The detection of individual pesticides was carried out accordingto the above procedure. Differential-pulse voltammograms (DPV)shows that the peak current decreased with increasing concentra-tion of pesticides and their calibration plots based on the depen-dence of the % inhibition on concentration were linear and wasshown in Fig. 3B and C for ML and AP, respectively. Fig. 3D and Eshows the effect of incubation time on residual enzyme activityat different known concentrations of ML and AP, respectively.The percentage of residual enzyme activity decreased with increas-ing incubation time for both the pesticides. This is because thelonger is the incubation time, the interaction between inhibitorand enzyme is more that provides greater inhibition. A completeinhibition was observed at 4 min incubation time with the1.3 ppm of ML and 0.85 ppm of AP. Limit of detection (LOD) andlimit of quantification (LOQ) calculations were carried out by usingthe following expressions (Madhusudana Reddy & Reddy, 2004;Madhusudana Reddy, Sreedhar, & Reddy, 2003).

LOD ¼ 3 Sb=S ð6Þ

LOQ ¼ 10 Sb=S ð7Þ

Where Sb is the standard deviation of mean values for ten differ-ential-pulse voltammograms of blank solution, S is the slope of theworking curve. A good linear relationship was obtained in therange from 0.07–1.3 ppm for ML and 0.1–0.85 ppm for AP (insetof Fig. 3B and C). The regression equations of I % = 0.0601C(%/ppm) + 26.2% (r = 0.9899, S.D. = 5.1834) and I % = 0.0756C(%/ppm) + 36.64% (r = 0.9990, S.D. = 1.1150) was obtained for MLand AP, respectively. The LOD values were 0.058 ppm, 0.044 ppmand LOQ values were 0.194 ppm, 0.147 ppm for ML and AP, respec-tively. The lowest LOD value is achieved in the case of AP, whichwas slightly more toxic than ML.

The robustness of the developed method was evaluated bystudying the concept of repeatability (new electrode, same stan-dard solution, same day, same analyst, ‘n’ is number of assays)and reproducibility (new electrode, new standard solution, differ-

ent days, different analyst, ‘n’ is number of assays) of the biosensortowards the inhibition of pesticides (Madhusudana Reddy & Reddy2004; Madhusudana Reddy et al., 2003). To study this experimentthe chosen concentration of the stock solutions of ML and AP were0.5 ppm. The repeatability and reproducibility values were studiedin terms of % RSD and were found to be as 2.59, 3.14 for AP and3.52, 4.19 for ML, respectively. The long term stability of the AChEbiosensor was investigated under the storage conditions (in coolplace), it was noticed that the activity of immobilised AChE wasstable up to one month.

3.5. Application towards the determination of pesticides in foodsamples

Organophosphates are some of the most widely used insecti-cides in fruit cultures. The pesticides were determined in fruitssamples without any previous extraction, clean up or preconcen-tration steps. Different inhibition values were observed dependingon the matrix analysed. However, the fruit samples showed an ini-tial matrix effect. We have noticed slight initial matrix effect forfruit samples due to denaturing conditions (pH) or presence ofascorbic acid (Hildebrandt, Bragos, Lacorte, & Marty, 2008b). Thematrix effect was minimised by measuring the difference in re-sponse between a spiked sample and blank of similar matrix.Table 1A shows differences from 12.5% to 53.5% which were ob-tained depending upon the matrix. The higher is the differencethe lower is the matrix effect, as observed for banana. High matrixeffects were observed for orange giving a difference of only 12.5%.The difference between blank values and spiked values for the restof the samples has given values higher than 30%. The biosensor wasable to find the pesticide residues such as ML and AP in the fruitsamples. We have observed through the Fig. 4A and B, that the per-centage of inhibition of ML and AP were found to be lower in com-parison with the sum of the spiked pesticide and matrix effect ofindividual samples. All measurements were performed in tripli-cate. The % inhibition of AChE for the spiked apples sample was

Table 1BComparison of different electrochemical/other techniques for the determination of AP and ML.

Biosensor Analyte (linear conc.range)

Technique (incubationtime)

Limit of detection(LOD)

Refs.

AChE–AuNPsa–CaCO3b–Auc–SiSGd ML (0.1–100 nM) CVm (10 min) 0.1 nM Narang, and Pundir (2011a)

AChE–Pin5COOHe–ZnSf/Auc ML (0.1–50 nM) SWVn (10 min) 0.1 nM Chauhan, Narang, and Pundir (2011b)AChE-Fe3O4NPg-MWCNTsh/Auc ML (0.1–40 nM) Amperometry (10 min) 0.1 nM Chauhan and Pundir (2011c)AChE–PAni–PPyj–MWCNTsh/GCEk ML (0.01–25 lg/mL) CVm (15 min) 1 ng/mL Du, Ye, Cai, Liu, and Zhang (2010)AChE-CHITl–AuNPsa–Auc ML (0.1–20 ng/mL) LSVo (15 min) 0.03 lg/L Du, Ding, Tao, and Chen (2008)

AP (7.5–500 ng/mL) lLC-FPDp 2.8 ng/mL Hooijschuur, Kientz, Dijksman, and Brinkman (2001)AP (5–140 ng/mL) ciELISAq 2 ng/mL Lee, Chang Ahn, Stoutamire, Gee, and Hammock (2003)

AChE–SiSGd–CPE ML (0.07–1.3 ppm) DPVr (4 min) 0.058 ppm Present workAP (0.1–0.85 ppm) DPVr(4 min) 0.044 ppm Present work

a AuNPs – Au nanoparticles.b CaCO3 – calcium carbonate.c Au – gold electrode.d SiSG – silica sol–gel.e Pin5COOH – poly (indole-5-carboxylic acid).f ZnS – zinc sulphide.g Fe3O4NP – iron oxide nanoparticle.h MWCNTs – multiwalled carbon nanotubes.i PAn – polyaniline.j PPy – polypyrrole.k GCE – glassy carbon electrode.l CHIT – chitosan.

m CV – cyclic voltammetry.n SWV – square wave voltammetry.o LSV – linear sweep voltammetry.p plLC-FPD – microcolumn liquid chromatography-flame photometric detector.q ciELISA – competitive indirect enzyme-linked immunosorbent assay.r DPV – differential pulse voltammetry.

194 P. Raghu et al. / Food Chemistry 142 (2014) 188–196

compared with the reference method i.e., Molecularly ImprintedMatrix Solid Phase Dispersion–Gas Chromatography (MIMSPD–GC) (Wang, Qiao, Ma, Zhao, & Xu, 2013) and was found to be ingood agreement.

The recovery efficiencies (R %) for the different systems werecalculated using the following equation.

% R ¼ 100 ð½pesticide� found=½pesticide� expectedÞ ð8Þ

The precision and accuracy of methodologies were tested withdifferent standard solutions of each pesticide and the relative stan-dard deviations (RSD) were calculated using the followingequation.

RSD ¼ Sb=X ð9Þ

Where Sb is standard deviation of mean inhibition values ob-tained and X is the mean inhibition (Pedrosa, Caetano, Machado,& Bertotti, 2008). The detection limit of various electroanalytical/other methods proposed for the determination of ML and AP wascompared with the present method and shown in Table 1B.

4. Conclusion

AChE biosensor has the ability for the direct detection of ML andAP in food samples and it requires no extraction or preconcentra-tion steps. The present study demonstrates the simple sol–gelentrapment procedure for the development of AChE biosensor,which requires less amount of enzyme. This biosensor was highlysensitive to the pesticide determination and it operates at a rela-tively low cost. In addition, the developed procedure can be em-ployed for the monitoring of different types of organic pollutantsand thus enlarging the future applicability of the biosensor.

Acknowledgements

The authors are very much thankful to the Department of Sci-ence and Technology (DST), Government of India, New Delhi forthe funding given through project no SR/FT/CS-025/2009.

References

Andreescu, S., Barthelmebs, L., & Marty, J. L. (2002). Immobilization ofacetylcholinesterase on screen-printed electrodes: Comparative studybetween three immobilization methods and applications to the detection oforganophosphorus insecticides. Analytica Chimica Acta, 464, 171–180.

Anitha, K., Venkata Mohan, S., & Jayarama Reddy, S. (2004). Development ofacetylcholinesterase silica sol–gel immobilized biosensor—An applicationtowards oxydemeton methyl detection. Biosensors and Bioelectronics, 20,848–856.

Aoki, K., Akimoto, K., Tokuda, K., Matsuda, H., & Osteryoung, J. (1984). Linear sweepvoltammetry at very small stationary disk electrodes. Journal of ElectroanalyticalChemistry, 171, 219–230.

Bard, A. J., & Faulkner, L. R. (2000). Electrochemical methods: Fundamentals andapplications. New York: Wiley.

Capitan-Valley, L. F., Deheidel, M. K. A., & Avivad, R. (1998). Determination ofcarbaryl in foods by solid-phase room-temperature phosphorimetry. Fresenius’Journal of Analytical Chemistry, 362, 307–312.

Chandra, U., Kumara Swamy, B. E., Gilbert, Ongera, & Sherigara, B. S. (2010).Voltammetric resolution of dopamine in the presence of ascorbic acid and uricacid at poly (calmagite) film coated carbon paste electrode. Electrochimica Acta,55, 7166–7174.

Chauhan, N., Narang, J., & Pundir, C. S. (2011a). Immobilization of rat brainacetylcholinesterase on porous gold-nanoparticle–CaCO3 hybrid materialmodified Au electrode for detection of organophosphorous insecticides.International Journal of Biological Macromolecules, 49, 923–929.

Chauhan, N., Narang, J., & Pundir, C. S. (2011b). Immobilization of rat brainacetylcholinesterase on ZnS and poly (indole-5-carboxylic acid) modified Auelectrode for detection of organophosphorus insecticides. Biosensors andBioelectronics, 29, 82–88.

Chauhan, N., & Pundir, C. S. (2011c). An amperometric biosensor based onacetylcholinesterase immobilized onto iron oxide nanoparticles/multi-walledcarbon nanotubes modified gold electrode for measurement oforganophosphorus insecticides. Analytica Chimica Acta, 701, 66–74.

Chithravathi, S., Kumara Swamy, B. E., Cahndra, U., Mamatha, G. P., & Sherigara, B. S.(2010). Electrocatalytic oxidation of sodium levothyroxine with phenylhydrazine as a mediator at carbon paste electrode: A cyclic voltammetricstudy. Journal of Electroanalytical Chemistry, 645, 10–15.

Daghbouche, Y., Garrigues, S., & Guardia, M. (1995). Solid phase preconcentration-Fourier transform infrared spectrometric determination of carbaryl and 1-naphthol. Analytica Chimica Acta, 314, 203–212.

De kok, A., & Hiemstra, M. (1992). Journal of Association of Official Analytical Chemists,75, 1063.

Du, D., Ding, J., Tao, Y., & Chen, X. (2008). Application of chemisorption/desorptionprocess of thiocholine for pesticide detection based on acetylcholinesterasebiosensor. Sensors and Actuators B, 134, 908–912.

Du, D., Ye, X., Cai, J., Liu, J., & Zhang, A. (2010). Acetylcholinesterase biosensor designbased on carbon nanotube-encapsulated polypyrrole and polyanilinecopolymer for amperometric detection of organophosphates. Biosensors andBioelectronics, 25, 2503–2508.

Gilbert, O., Kumara Swamy, B. E., Chandra, U., & Sherigara, B. S. (2009).Simultaneous detection of dopamine and ascorbic acid using polyglycinemodified carbon paste electrode: A cyclic voltammetric study. Journal ofElectroanalytical Chemistry, 636, 80–85.

Hildebrandt, A., Bragos, R., Lacorte, S., & Marty, J. L. (2008b). Performance of aportable biosensor for the analysis of organophosphorus and carbamateinsecticides in water and food. Sensors and Actuators B, 133, 195–201.

Hildebrandt, A., Ribas, J., Bragos, R., Marty, J. L., Tresanchez, M., & Lacorte, S. (2008a).Development of a portable biosensor for screening neurotoxic agents in watersamples. Talanta, 75, 1208–1213.

Hooijschuur, E. W. J., Kientz, Ch. E., Dijksman, J., & Brinkman, U. A. T. (2001).Potential of microcolumn liquid chromatography and capillary electrophoresiswith flame photometric detection for determination of polar phosphorus-containing pesticides. Chromatographia, 54, 295–301.

Kindervater, R., Kunnecke, W., & Schmid, R. D. (1990). Exchangeable immobilizedenzyme reactor for enzyme inhibition tests in flow-injection analysis using amagnetic device. Determination of pesticides in drinking water. AnalyticaChimica Acta, 234, 113–117.

Lee, J. K., Chang Ahn, K., Stoutamire, D. W., Gee, S. J., & Hammock, B. D. (2003).Development of an enzyme-linked immunosorbent assay for the detection ofthe organophosphorus insecticide Acephate. Journal of Agricultural and FoodChemistry, 51, 3695–3703.

Leon-Gonzales, M. F., & Townshend, A. (1991). Flow-injection determination ofparaoxon by inhibition of immobilized acetylcholinesterase. Analytica ChimicaActa, 236, 267–272.

Li, J., Chia, L. S., Goh, N. K., & Tan, S. N. (1998). Silica sol–gel immobilizedamperometric biosensor for the determination of phenolic compounds.Analytica Chimica Acta, 362, 203–211.

Madhusudana Reddy, T., & Reddy, S. J. (2004). Differential pulse adsorptive strippingvoltammetric determination of nifedipine and nimodipine in pharmaceuticalformulations, urine, and serum samples by using a clay-modified carbon-pasteelectrode. Analytical Letters, 37, 2079–2098.

Madhusudana Reddy, T., Sreedhar, M., & Reddy, S. J. (2003). Voltammetric behaviorof cefixime and cefpodoxime proxetil and determination in pharmaceuticalformulations and urine. Journal of Pharmaceutical and Biomedical Analysis, 31,811–818.

Maria Dolores, W., Ana, A., Amadeo, R. F. A., Luis, P., & Mariano, C. (2001). Gaschromatographic determination of pesticides in vegetable samples bysequential positive and negative chemical ionization and tandem massspectrometric fragmentation using an ion trap analyser. Analyst, 126, 46–51.

Martorell, D., Cespedes, F., Martinez-Fabregas, E., & Alegret, S. (1994).Amperometric determination of pesticides using a biosensor based on apolishable graphie-epoxy biocomposite. Analytica Chimica Acta, 290, 343–348.

Mc Garvey, B. D. (1993). High-performance liquid chromatographic methods for thedetermination of N-methylcarbamate pesticides in water, soil, plants and air.Journal of Chromatography A, 642, 89–105.

Mohammadi, H., Amine, A., Cosnier, S., & Mousty, C. (2005). Mercury–enzymeinhibition assays with an amperometric sucrose biosensor based on atrienzymatic-clay matrix. Analytica Chimica Acta, 543, 143–149.

Murillo Pulgarin, J. A., Molina, A. A., & Fernandez Lopez, P. (2006). Automaticchemiluminescence-based determination of carbaryl in various types ofmatrices. Talanta, 68, 586–593.

Newsome, W. H., Lau, B. P. Y., Ducharme, D., & Lewis, D. (1995). Journal of Associationof Official Analytical Chemists, 78, 1312–1321.

Pandey, P. C., & Singh, G. (2001). Tetraphenylborate doped polyaniline based novelpH sensor and solid-state urea biosensor. Talanta, 55, 773–782.

Pandey, P. C., Upadhyay, S., Tiwari, I., Singh, G., & Tripathi, V. S. (2001). A novelferrocene encapsulated palladium-linked ormosil-based electrocatalyticdopamine biosensor. Sensors and Actuators B, 75, 48–55.

Park, T. M. (1999). Amperometric determination of hydrogen peroxide by utilizing asol–gel – Derived biosensor incorporating an osmium redox polumer asmediator. Analytical Letters, 32, 287–298.

Park, T. M., Lwuoha, E. I., Smyth, M. R., Freoney, R., & Mc Shane, A. J. (1997). Sol–gelbased amperometric biosensor incorporating an osmium redox polymer asmediator for detection of L-lactate. Talanta, 44, 973–978.

Pedrosa, V. A., Caetano, J., Machado, S. A. S., & Bertotti, M. (2008). Determination ofparathion and carbaryl pesticides in water and food samples using a selfassembled monolayer/acetylcholinesterase electrochemical biosensor. Sensors,8, 4600–4610.

P. Raghu et al. / Food Chemistry 142 (2014) 188–196 195

Rodrignez, L. A., Monferrer-Pons, L., Esteve Romero, J. S., Garcia Alvarez, C. M. C., &Ramis Rames, G. (1997). Spectrophotometric determination of carbamatepesticides with diazotized trimethylaniline in a micellar medium of sodiumdodecyl sulfate. Analyst, 122, 459–463.

Rouillon, R., Mionelto, N., & Marty, J. L. (1992). Acetylcholine biosensor involvingentrapment of two enzymes. Optimization of operational and storageconditions. Analytica Chimica Acta, 268, 347–350.

Sampath, S., & Lev, O. (1996). Inert metal-modified, composite ceramic-carbon,amperometric biosensors: Renewable, controlled reactive layer. AnalyticalChemistry, 68, 2015–2021.

Shane, D., Mousty, C., & Cosnie, S. (2004). Subnanomolar cyanide detection atpolyphenol oxidase/clay biosensors. Analytical Chemistry, 76, 178–183.

Skaladal, P. (1992). Detection of organophosphate and carbamate pesticides usingdisposable biosensors based on chemically modified electrodes andimmobilized cholinesterase. Analytica Chimica Acta, 269, 281–287.

Suwansa-ard, S., Kanatharana, P., Asawatreratanakul, P., Limsakul, C.,Wongkittisuka, B., & Thavareingkul, P. (2005). Semi disposable reactor

biosensors for detecting carbamate pesticides in water. Biosensors andBioelectronics, 21, 445–454.

Tuxelli, A., Bag, H., & Turker, A. R. (2001). Spectrophotometric determination ofsome pesticides in water samples after preconcentration with Saccharomycescerevisiae immobilized on sepiolite. Fresenius’ Journal of Analytical Chemistry,371, 1134–1138.

Wang, X., Qiao, X., Ma, Y., Zhao, T., & Xu, Z. (2013). Simultaneous determinationof nine trace organophosphorous pesticide residues in fruit samples usingmolecularly imprinted matrix solid-phase dispersion followed bygas chromatography. Journal of Agricultural and Food Chemistry, 61,3821–3827.

Wang, J. H., Wang, G. T., & Yuan, S. M. (1999). Chinese Journal of Analytical Laboratory(Fenxishiyanshi), 18, 55.

Yao, T., & Takashima, K. (1998). Amperometric biosensor with a compositemembrane of sol–gel derived enzyme film and electrochemically generatedpoly (1,2-diaminobenzene) film. Biosensors and Bioelectronics, 13, 67–73.

196 P. Raghu et al. / Food Chemistry 142 (2014) 188–196

Enzyme and Microbial Technology 52 (2013) 377– 385

Contents lists available at SciVerse ScienceDirect

Enzyme and Microbial Technology

jou rn al h om epage: www.elsev ier .com/ locate /emt

A novel electrochemical biosensor based on horseradish peroxidase immobilizedon Ag-nanoparticles/poly(l-arginine) modified carbon paste electrode towardthe determination of pyrogallol/hydroquinone

P. Raghua, T. Madhusudana Reddya,∗, K. Reddaiaha, L.R. Jaidevb, G. Narasimhab

a Electrochemical Research Laboratory, Department of Chemistry, S.V.U. College of Sciences, Sri Venkateswara University, Tirupati 517502, Andhra Pradesh, Indiab Applied Microbiology Laboratory, Department of Virology, Sri Venkateswara University, Tirupati 517502, Andhra Pradesh, India

a r t i c l e i n f o

Article history:Received 28 January 2013Received in revised form 21 February 2013Accepted 24 February 2013

Keywords:Horseradish peroxidaseSilica sol–gelPyrogallolHydroquinoneCarbon paste electrodeSilver nanoparticle

a b s t r a c t

A novel electrochemical biosensor for the determination of pyrogallol (PG) and hydroquinone (HQ) hasbeen constructed based on the poly l-arginine (poly(l-Arg))/carbon paste electrode (CPE) immobilizedwith horseradish peroxidase (HRP) and silver nanoparticles (AgNPs) through the silica sol–gel (SiSG)entrapment. The electrochemical properties of the biosensor were characterized by employing the elec-trochemical techniques. The proposed biosensor showed a high sensitivity and fast response toward thedetermination of PG and HQ around 0.18 V. Under the optimized conditions, the anodic peak current ofPG and HQ was linear with the concentration range of 8 �M to 30 × 10−5 M and 1–150 �M. The limit ofdetection (LOD) and limit of quantification (LOQ) were found to be 6.2 �M, 20 �M for PG and 0.57 �M,1.92 �M for HQ respectively. The electrochemical impedance spectroscopy (EIS) studies have confirmedthat the occurrence of electron transfer at HRP-SiSG/AgNPs/poly(l-Arg)/CPE was faster. Moreover thestability, reproducibility and repeatability of the biosensor were also studied. The proposed biosensorwas successfully applied for the determination of PG and HQ in real samples and the results were foundto be satisfactory.

© 2013 Elsevier Inc. All rights reserved.

1. Introduction

Pyrogallol (PG) and hydroquinone (HQ) are derivatives of phe-nolic compounds which are important contaminants in medicalfood and environmental matrices. Reliable analytical proceduresare required for the determination of PG and HQ in variousmatrices with high sensitivity. So far, many methods have beendeveloped for their determination, including liquid chromatogra-phy [1,2], synchronous fluorescence [3], chemiluminescence [4,5],spectrophotometry [6], gas chromatography/mass spectrometry[7], pH based-flow injection analysis [8], electrochemical meth-ods [9,10], etc., However, most of the above methods have somedisadvantages, such as time consuming, high cost, low sensitivityand complicate pretreatment. In recent past, more attention on thedevelopment of biosensor was made due to its advantages suchas easy preparation, fast detection, low consume of time and highsensitivity [11].

Horseradish peroxidase (HRP) is an important enzyme and isalways used as an electron acceptor. Among peroxidases, HRP has

∗ Corresponding author. Tel.: +91 877 2289303.E-mail addresses: tmsreddysvu@gmail.com,

tmsreddy svu@yahoo.co.in (T. Madhusudana Reddy).

been one of the most widely studied enzymes in the developmentof enzyme based biosensor. Because of the deep embedding of theHRP-active site, which is in unfavorable orientation [12], it is a chal-lenging task to obtain the direct electrochemistry of HRP. Accordingto Marcus theory, the electron transfer (ET) distance is a deci-sive factor for the direct electrochemistry of redox enzyme, whichdepends on the overall distance between the redox site withinthe enzyme and the electrode surface, and the orientation of theenzyme on the electrode [13]. In order to prepare good biosensors,many materials such as nanoparticles, redox dyes, conducting poly-mers, biomolecules and ionic liquids were employed to improve themicroenvironment around the enzyme to provide suitable orienta-tion and to accelerate the electron transfer between the enzymeand the surface of the electrode [11].

Noble metal nanoparticles have been extensively used in thedesigning and in construction of enzyme biosensors due to theirunique characteristics, such as high surface energy and surface tovolume ratio, ability to decrease proteins–metal particle distance,good mechanical, thermal and chemical stability [14,15]. So far,the direct electron transfer of HRP has been reported at nano-material surfaces such as gold nanoparticles (AuNPs) [16], goldnanowire array electrodes [17], cadmium sulphide [CdS] nanorods[18], and carbon nanotubes (CNTs) [19,20]. The stability and sen-sitivity of a biosensor could be improved by choosing suitable

0141-0229/$ – see front matter © 2013 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.enzmictec.2013.02.010

378 P. Raghu et al. / Enzyme and Microbial Technology 52 (2013) 377– 385

enzyme immobilization matrix and by adapting better enzymeimmobilization strategies. Recently several valuable immobiliza-tion strategies have been employed including absorption [21],cross-linking [22], layer-by-layer assembly [23], sol–gel entrap-ment [24], electropolymerization [25,26], etc., Among those silicasol–gel (SiSG) entrapment technology has attracted much attentionin the field of immobilization of a variety of biomolecules, becauseof its special features such as chemical inertness, physical rigid-ity, high-thermal stability, biodegradation and optical transparency[27,28]. However, these HRP-based biosensors require mediators totransfer electrons between the electrode and HRP.

In our previous work, we have developed a biosensor basedon the CPE immobilized with HRP, through SiSG entrapment forthe determination of hydroquinone (HQ). The electrochemicalproperties of biosensor were characterized by employing the elec-trochemical methods like cyclic voltammetry (CV) and differentialpulse voltammetry (DPV). The electrocatalytic response of HQ wasdetected in methanol, ethanol, 2-propoanol, 1-butanol and ace-tone. The good results were obtained in ethanol as a solvent andacetate buffer solution (ABS) as supporting electrolyte, the exper-iments were carried out in combination of these two media. Theelectrochemical impedance spectroscopy (EIS) result confirmed theoccurrence of rapid electron transfer at HRP-SiSG/CPE. The pro-posed sensor was successfully applied for the determination of HQin real samples and the result were found to be commensurate[29].

In this present work, we have concentrated on embeddingHRP into the network of SiSG/AgNPs/poly(l-Arg)/CPE. This tech-nology exhibited a remarkable advantage of AgNPs/poly(l-Arg) andSiSG network. HRP was effectively embedded into the network ofAgNPs/poly(l-Arg) which in turn can promote the direct electrontransfer of the enzyme immobilized on the electrode surface. Theelectrocatalytic behavior of this biosensor has also been investi-gated in detail. The resulting biosensor exhibited high sensitivityand good stability.

2. Experimental

2.1. Reagents

All chemicals were obtained from commercial sources and used without fur-ther purification. Horseradish peroxidase (E.C. 1.11.1.7 type-VI-A-S/5 mg, Amoraciarusticana source, 1840 U/mg), pyrogallol and hydroquinone were purchased fromSigma–Aldrich chemicals Co., USA. Tetraethyl orthosilicate (TEOS), cetyltrimethylammonium bromide (CTAB), Triton-X-100 were obtained from Sigma–Aldrichchemicals Co., USA. The graphite fine powder was procured from Lobo Chemieand silicon oil from Himedia. Silver nanoparticles (AgNPs) used in the presentstudy were synthesized from fungal culture Aspergillis niger and their size (1–3 nm)and shape (spherical) were characterized according to Jaidev and Narasimha [30].Acetate buffer solution (ABS) was prepared by mixing 0.1 M sodium acetate and

0.1 M acetic acid. All the aqueous solutions were prepared with double distilledwater. The enzyme stock solution and working solutions of chemicals were storedin cool place.

2.2. Apparatus

The electrochemical measurements were conducted in a three electrodes cellat a room temperature 25 ± 2 ◦C. The working electrode was a enzyme immobilizedcarbon paste electrode (HRP-SiSG/AgNPs/poly(l-Arg)/CPE). The reference electrodewas a saturated calomel electrode system and glassy carbon rod electrode was usedas an auxiliary electrode. Electrochemical measurements were carried out usingCH-Electrochemical Analyzer (Model CHI-660D, CH Instruments, USA).

2.3. Preparation of poly(l-arginine) modified carbon paste electrode(poly(l-Arg)/CPE)

The carbon past electrode was prepared by hand mixing 85% graphite powderand 15% silicon oil in an agate mortar. The carbon paste was then packed into thecavity of a homemade carbon paste electrode with a diameter of 2 mm and smoothedon a weighing paper [31,32]. The 0.038 M of aqueous solution of l-arginine wasplaced in the electrochemical cell and dipped with CPE and was scanned for eightmultiple cycles between the potential ranges from −0.6 V to +1.6 V at 100 mV/s. Afterpolymerization, the poly(l-Arg) film was rinsed sufficiently with double distilledwater.

2.4. Fabrication of HRP-SiSG/AgNPs/poly(l-Arg)/CPE

A homogenous TEOS silica sol–gel was prepared by mixing 2 ml of TEOS, 1 ml ofH2O, 50 �l of 0.1 M KCl, 25 �l of 10% Triton-X-100. The mixture was stirred for 1 hfor obtaining clear sol. The sol can be stored for about one month when it was keptin refrigerator.

The 5 �l of 5 mg/ml enzyme stock solution was added to the mixture of 5 �lof stock SiSG solution, 40 �l of ABS and 10 �l of AgNPs. A drop of this dispersionwith a volume of 5 �l was cast onto the surface of the poly(l-Arg)/CPE, then it wasallowed to polymerize at room temperature for 3–5 min. The electrode was gentlywashed with ABS and was used for further experimental procedure [33]. The 2.5 Uof enzyme was immobilized on the electrode surface. The fabrication procedure ofthe biosensor is illustrated in Scheme 1.

3. Results and discussion

3.1. Electrochemical polymerization of l-arginine on carbonpaste electrode

l-Arginine is an amino acid and its electrochemical polymeriza-tion potential was between +1.6 V to −0.6 V. The potential windowscan lies in the positive direction and this was the most importantfactor in preparing the poly(l-Arg) film. If the potential windowwas less than 1.6 V or greater than −0.6 V, it was observed thatthe formation of poly film on the CPE was not stable. On the otherhand, a stable poly film was obtained by the electropolymerizationbetween the potential windows of −0.6 V to +1.6 V and with a max-imum of eight cycles on CPE. Fig. 1A shows the growth of polymerfilm of 0.038 M aqueous l-Arginine solution on the surface of carbon

Scheme 1. A schematic diagram showing the steps involved in the fabrication of HRP-SiSG/AgNPs/poly(l-Arg)/CPE with reaction mechanism.

P. Raghu et al. / Enzyme and Microbial Technology 52 (2013) 377– 385 379

Fig. 1. (A) Cyclic voltammogram for the electrochemical polymerization of (l-arginine) at the carbon paste electrode, inset: structure of l-arginine. SEM imagesof (B) CPE and (C) HRP-SiSG/AgNPs/poly(l-Arg)/CPE films captured at 3 �M magni-fications.

paste electrode. During the process of multiple cycles the voltam-mogram has gradually descended with increase of cycle time. Thisindicates that the poly(l-Arg) film was formed and deposited onthe surface of carbon paste electrode [34].

3.2. Surface morphological characterizations using scanningelectron microscopy (SEM)

SEM study was employed to investigate the surface morphol-ogy of bare CPE and HRP-SiSG/AgNPs/poly(l-Arg)/CPE films. Thesurface of bare carbon paste electrode (bare CPE) was irregu-larly shaped with 3 �m (3K×) magnification and is shown inFig. 1B. In this image the flakes of graphite are evenly distributed

Fig. 2. Cyclic voltammograms of CPE with buffer (a), CPE (b), HRP-SiSG/CPE (c), HRP-SiSG/AgNPs/CPE (d) and HRP-SiSG/AgNPs/poly(l-Arg)/CPE (e) in 1 mM PG/0.1 M ABS(pH 5.0) (A) or in 1 mM HQ/0.1 M ABS (pH 4.5) (B) at a scan rate of 25 mV/s.

throughout the film surface. Fig. 1C shows the SEM image of HRP-SiSG/AgNPs/poly(l-Arg)/CPE with magnification of 3 �m (3K×).Unlike bare CPE film, the enzyme modified CPE film surface possesseveral bright globular structures, along with uniform and gummynature background. The SEM results confirmed the formation ofHRP-SiSG/AgNPs/poly(l-Arg)/CPE film and clearly demonstratedthat AgNPs modified electrode surface is more effective for HRPimmobilization.

3.3. Investigation of electrochemical behavior of various filmmodified CPE’s using cyclic voltammetric (CV) studies

The electrochemical behaviors of PG and HQ at the bare CPE,HRP-SiSG/CPE, HRP-SiSG/AgNPs/CPE and HRP-SiSG/AgNPs/poly(l-Arg)/CPE in 0.1 M ABS (pH 5.0/4.5) were studied using CV. Fig. 2Ashows the electrochemical responses obtained for the various filmmodified CPEs in 0.1 M ABS (pH 5.0) with and without 0.1 mM PG.As shown in Fig. 2A no peak was observed at the bare CPE withoutPG (curve ‘a’). When the 0.1 mM PG solution was added, the anodicand cathodic peaks of PG was observed at 0.228 V and 0.069 V atbare CPE (curve ‘b’), respectively. The peak potential separation(�Ep) was about 0.159 V, which indicates that the PG exhibitsa quasireversible electrochemical behavior at bare CPE. While atHRP-SiSG/CPE (curve ‘c’) and HRP-SiSG/AgNPs/CPE (curve ‘d’), thepeak currents of both anodic and cathodic peaks slightly increases.When it is modified with poly arginine (curve ‘e’), there was a goodagreement of increase in both anodic and cathodic peak currents,the anodic and cathodic peaks appeared at 0.162 V and 0.079 Vrespectively with peak to peak separation of 0.083 V. Apart fromthis, the anodic peak potential decreased from 0.228 to 0.162 V and

380 P. Raghu et al. / Enzyme and Microbial Technology 52 (2013) 377– 385

the cathodic peak potentials shifted from 0.069 to 0.079 V. Theseresults indicate that the modifiers provide a mimic environment forthe functioning of proteins, in which the native conformations ofthe proteins are retained and the electron transfer rates are greatlyenhanced compared with those involving proteins alone at bareelectrode [35].

The CV behavior of HQ was also studied at various film mod-ified CPEs under the same conditions, as shown in Fig. 2B. AtHRP-SiSG/CPE (curve ‘c’), the anodic and cathodic peak potentialof HQ appears at 0.255 V and 0.027 V, respectively, with a large�Ep of 0.228 V. While at HRP-SiSG/AgNPs/poly(l-Arg)/CPE (curve‘e’), the anodic and cathodic peaks appeared at 0.177 and 0.064 V,respectively, with a great reduced in the �Ep value to 0.113 V. Inaddition, the anodic and cathodic peak currents were also greatlyenhanced. The surface concentration of electroactive HRP (� ) inHRP-SiSG/AgNPs/poly(l-Arg)/CPE surface was estimated accordingto following equation [36].

Ip = n2F2A��

4RT(1)

where Ip is the peak current, A is the electrode active surface area,� is the scan rate, n is the number of electrons, R, T and F have theirusual meanings. By considering the above values, � was calculatedas 8.63 × 10−8 mol cm−2, which was found to be about 45 timesmore than the monolayer coverage of HRP (5 × 10−11 mol cm−2)on 3-mercaptopropionic acid modified gold electrode [37]. Theseresults indicated that the multiple layers of active HRP were coatedon the electrode surface. The AgNPs/poly(l-Arg)/CPE/SiSG matrixwas more efficient for HRP immobilization. The surface concen-tration of electroactive HRP (� ) with different immobilizationmatrices was compared with the present immobilization matrixin Table 1A.

3.4. Effect of solution pH

Fig. 3A and B shows the effect of pH on the electrochemi-cal response of the HRP-SiSG/AgNPs/poly(l-Arg)/CPE toward thedetermination of PG and HQ respectively. A well defined quasi-reversible redox peaks corresponding to Fe(III/II) redox process of

HRP and PG/HQ was observed in the pH range varying from 3.5to 6.5. The redox peak currents increased with increase in pH ofthe ABS up to 5.0/4.5 for PG/HQ and thereafter decreased grad-ually. From these results pH 5.0/4.5 for PG/HQ were selected asthe optimum pH values for subsequent experiments. The peakpotentials decreases linearly with the increase of pH value and thesame is shown in the inset of Fig. 3A and B for PG and HQ respec-tively. The regression equations between the potential (E) and pHwas E (V) = −0.07 pH + 0.5205 (r = 0.9961) for PG and E (V) = −0.052pH + 0.3694 (r = 0.9798) for HQ. The slopes of potential (E) vs. pHwere −70 mV/pH for PG and −52 mV/pH for HQ. These slope val-ues were close to the theoretical slope of (59 mV/pH), which is inaccordance with Nernst equation for transfer of equal number ofelectrons and protons in the reaction [31,32].

3.5. Influence of scan rate

The cyclic voltammograms (CVs) corresponding to various scanrates were also investigated for the fabricated biosensor. Both theanodic and cathodic peak currents increased with increasing poten-tial scan rate. As shown in Fig. 3C and D, a well characterizedredox peaks were observed for PG and HQ respectively. Moreover,it was found from the inset of Fig. 3C and D, that the peak currentswere proportional to the square root of scan rates, suggesting atypical the semi-infinite linear diffusion-controlled electrochem-ical behavior feature for both PG and HQ. The catalytic processof HRP-SiSG/AgNPs/poly(l-Arg)/CPE surface toward PG/HQ can beexpressed as follows [41].

HRP (Fe)II + C6H7O3/C6H6O2

→ Compound I ([FeIV O]+) + C6H5O3/C6H4O2 (R1)

Compound I ([FeIV O]+) + e− + H+ → Compound II ([FeIV O])

(R2)

Compound II ([FeIV O]) + e− + H+ → HRP (FeIII) + H2O (R3)

Table 1AMichaelis–Menten constant and � values for HRP in different biosensors.

Biosensor � a Kappm

b Refs.

HRP/MPAc/Aud 5 × 10−11 mol cm−2 [37]HRP/nano-Au/che/GCEf 1.2 × 10−9 mol cm−2 1.55 mM [38]HRP/DPPAg/PGEh 1.5 × 10−10 mol cm−2 [39]HRP/colloidal Au/CPEi 7.5 × 10−11 mol cm−2 3.69 mM [40]HRP/nano-Au/Cysj/SiSGk/Au 1.1 mM [46]HRP/colloidal Au/SPCEl 1.3 mM [47]HRP/MBm/MWNTn/GCE 3.2 × 10−12 mol cm−2 0.12 mM [48]CPE/sol–gel-Fero/HRP/sol–gel 0.19 mM [49]HRP-SiSG/AgNPsp/poly(l-Arg)q/CPE 8.63 × 10−8 mol cm−2 0.25 mM (for PG)0.11 mM (for HQ) Present work

a � , electroactive concentration of HRP on the immobilized electrode surface.b Kapp

m , apparent Michaelis–Menten constant.c MPA, 3-mercaptopropionic acid.d Au, gold electrode.e ch, choline.f GCE, glassy carbon electrode.g DPPA, dipalmitoylphosphatidic acid.h PGE, pyrolytic graphite electrode.i CPE, carbon paste electrode.j Cys, cysteine.k SiSG, silica sol–gel.l SPCE, screen printed carbon electrode.

m MB, methylene blue.n MWNT, multiwalled carbon nanotubes.o Fer, ferrocene.p AgNPs, silver nanoparticles.q Poly(l-Arg), poly(l-arginine).

P. Raghu et al. / Enzyme and Microbial Technology 52 (2013) 377– 385 381

Fig. 3. (A and B) The effect of pH on the redox behavior of HRP-SiSG/AgNPs/poly(l-Arg)/CPE in 0.1 M ABS solution with pH ranging from 3.5 to 6.5 for PG and HQ respectively.The inset is Ipa/Epa vs. pH plot. (C and D) Typical cyclic voltammograms of HRP-SiSG/AgNPs/poly(l-Arg)/CPE and the calibration plot of wave current at different scan ratesfor 1 mM of PG/0.1 M ABS pH (5.0) ((a) 10 mV/s, (b) 15 mV/s, (c) 20 mV/s, (d) 25 mV/s, (e) 30 mV/s, (f) 35 mV/s, (g) 40 mV/s, (h) 45 mV/s, (i) 50 mV/s, (j) 55 mV/s, (k) 60 mV/s,(l) 70 mV/s, (m) 80 mV/s, (n) 90 mV/s, (o) 100 mV/s) and 1 mM HQ/0.1 M ABS pH (4.5) ((a) 20 mV/s, (b) 25 mV/s, (c) 30 mV/s, (d) 35 mV/s, (e) 40 mV/s, (f) 45 mV/s, (g) 50 mV/s,(h) 55 mV/s, (i) 60 mV/s, (j) 65 mV/s, (k) 70 mV/s, (l) 80 mV/s, (m) 90 mV/s, (n) 100 mV/s, (o) 110 mV/s, (p) 120 mV/s). (E and F) Differential pulse voltammograms of (a)0.8 × 10−5 M, (b) 1 × 10−5 M, (c) 2 × 10−5 M, (d) 3 × 10−5 M, (e) 4 × 10−5 M, (f) 5 × 10−5 M, (g) 6 × 10−5 M, (h) 8 × 10−5 M, (i) 9 × 10−5 M, (j) 10 × 10−5 M, (k) 14 × 10−5 M, (l)18 × 10−5 M, (m) 22 × 10−5 M, (n) 26 × 10−5 M, (o) 30 × 10−5 M for PG in 0.1 M ABS (pH 5.0) and (a) 1 × 10−6 M, (b) 3 × 10−6 M, (c) 6 × 10−6 M, (d) 10 × 10−6 M, (e) 15 × 10−6 M,(f) 20 × 10−6 M, (g) 25 × 10−6 M, (h) 30 × 10−6 M, (i) 40 × 10−6 M, (j) 50 × 10−6 M, (k) 60 × 10−6 M, (l) 70 × 10−6 M, (m) 80 × 10−6 M, (n) 90 × 10−6 M, (o) 100 × 10−6 M, (p)150 × 10−6 M for HQ in 0.1 M ABS (pH 4.5). Inset (A and C) graph of peak current vs. the concentration of PG and HQ respectively. Inset (B and D) Lineweaver–Burk plots forPG and HQ respectively.

3.6. Electrocatalytic determination of PG and HQ at fabricatedbiosensor

The electrocatalytic activity of HRP-SiSG/AgNPs/poly(l-Arg)/CPE toward PG and HQ was investigated by employingdifferential pulse voltammetry (DPV) experiment. Fig. 3E andF shows DPV curves for different concentrations of PG/HQ in0.1 M ABS (pH 5.0/4.5). The results showed that the anodic peakcurrent was proportional to the concentration from 0.8 × 10−5

to 30 × 10−5 M and 1–150 �M for PG and HQ respectively. Agood linear relationship was obtained (inset A and C of Fig. 3Eand F) with the regression equations of Ipa (�A) = 0.3861 C(10−5 M l−1) + 1.65 (r = 0.9968) for PG and Ipa (�A) = 0.42 C(�M l−1) + 0.7056 (r = 0.9994) for HQ respectively. The LODand LOQ of this biosensor were estimated to be 6.2 �M and 20 �M

for PG, 0.57 �M and 1.92 �M for HQ respectively. The highersensitivity of the sensor may result from the good biocompatiblemicroenvironment around the enzyme [42]. The LOD and LOQvalues were calculated by using the following expressions [43,44].

LOD = 3Sb

S(2)

LOQ = 10Sb

S(3)

where Sb is the standard deviation of mean values for tendifferential-pulse voltammograms of blank solution, S is the slopeof the working curve.

The apparent Michaelis–Menten constant (Kappm ), gives an indi-

cation toward the enzyme–substrate kinetics for the biosensor,

382 P. Raghu et al. / Enzyme and Microbial Technology 52 (2013) 377– 385

Fig. 4. (A) EIS of (a) bare CPE, (b) HRP-SiSG/AgNPs/poly(l-Arg)/CPE, (c) bare Pt, (d)HRP-SiSG/AgNPs/poly(l-Arg)/Pt, (e) bare GCE, (f) HRP-SiSG/AgNPs/poly(l-Arg)/GCErecorded in 0.1 M KCl containing 1 mM K3[Fe(CN)6]/K4[Fe(CN)6]. Amplitude: 5 mV,frequency: 1 Hz to 100 kHz. (B) EIS of (a) HRP-SiSG/AgNPs/poly(l-Arg)/CPE, (b) bareCPE, (c) CPE/AgNPs, (d) CPE/AgNPs/poly(l-Arg) recorded in 0.1 M ABS pH (4.5)/1 mMK3[Fe(CN)6]/K4[Fe(CN)6]/2 �M of HQ. Amplitude: 5 mV, frequency: 1 Hz to 100 kHz.

and it was calculated from the electrochemical version of theLineweaver–Burk equation [45].

1iss

= 1imax

+ Kappm

imaxC(4)

where iss is the steady state current after the addition of substrateand imax is the maximum current measured under saturated sub-strate condition. The Kapp

m and imax values of the biosensor werefound to be 0.25 mM and 20 �A for PG and 0.11 mM and 10 �A forHQ respectively (inset B and D of Fig. 3E and F). Smaller is the Kapp

mvalue, higher is the enzyme activity (HRP) and it shows higher affin-ity toward PG and HQ. The Kapp

m value of the proposed biosensor wascompared with Kapp

m values of other biosensors in Table 1A.

3.7. Electrochemical impedance spectroscopy (EIS)characterization of biosensor

The EIS is a powerful analytical tool to characterize the elec-trochemical process occurring at the solution/electrode interface.The curve of the EIS consists of a semicircle portion and a linearportion. At the higher frequencies the semicircle portion wasobserved, which gives the information of impedance in the formof a diameter. A linear portion was observed at lower frequencies,which represents higher electron transfer. The electron transferrate at the surface of the electrode was influenced by the sur-face electron-transfer resistance (Ret) of the electrode. Fig. 4Ashows the curves of Nyquist plots (Z′′ vs. Z′) for different typesof electrodes. The electrochemical impedance measurementswere carried out in 1 mM K3[Fe(CN)6]/K4[Fe(CN)6] and 0.1 M KClsolution. The alternating voltage was 5 mV and the frequency

range was 1 Hz to 100 kHz. It was seen that the bare CPE (a) andHRP-SiSG/AgNPs/poly(l-Arg)/CPE (b) exhibited an almost straightlines, meaning that the probe found it easier to access the surfaceof the electrode. The Nyquist diameter for bare Pt electrode (c)and bare glassy carbon electrode (GCE) (e) was much largerthan that of the corresponding modified HRP-SiSG/AgNPs/poly(l-Arg)/Pt (d) and HRP-SiSG/AgNPs/poly(l-Arg)/GCE (f), whichindicates the modified electrodes shows less impedance andmore electron transfer rate. The order of impedance for differentelectrodes was as follows, bare GCE > HRP-SiSG/AgNPs/poly(l-Arg)/GCE > bare Pt > HRP-SiSG/AgNPs/poly(l-Arg)/Pt > bareCPE > HRP-SiSG/AgNPs/poly(l-Arg)/CPE.

Fig. 4B shows EIS results for the different assembly stagesof biosensor in 1 mM K3[Fe(CN)6]/K4[Fe(CN)6] solution pre-pared by 0.1 M ABS (pH 4.5) and 2 �M of HQ solution. TheRandles equivalence circuit model was employed to fit theobtained impedance data, which was shown in inset of Fig. 4B(where Rs represents the solution resistance, Cdl is the dou-ble layer capacitance, Ret is the electron transfer resistance,Rp is the polarization resistance and Q is the CPE (constantphase element)). The curves a–d in Fig. 4B express HRP-SiSG/AgNPs/poly(l-Arg)/CPE (a), bare CPE (b), CPE/AgNPs (c)and CPE/AgNPs/poly(l-Arg) (d) electrodes respectively. Accord-ing to the curves, ‘a’ has less impedance (straight line) and theremaining electrodes show slight curve nature. The electron trans-fer rate constant (Ket) values of various electrodes were, bare CPE(0.073 cm/s) < CPE/AgNPs (0.076 cm/s) < CPE/AgNPs/poly(l-Arg)(0.099 cm/s) < HRP-SiSG/AgNPs/poly(l-Arg)/CPE (0.12 cm/s). Fromthe above results it was noticed that the increase in electrontransfer rate constant corresponds to the decrease in impedance.

3.8. Stability and reproducibility of biosensor

The fabricated biosensor was continuously tested for 50 suc-cessive cycles in the potential range from −0.2 to 0.5 V at the scanrate of 50 mV/s in ABS (pH 4.5/5.0) containing 1 mM PG/HQ. It wasnoticed that after 50 cycles there was no disturbance in the peakpotentials of the system, whereas the peak currents of the systemreduced to 88% in comparison with the initial signal. This indi-cates that the assembly layer was stably fixed on the electrodesurface. The results obtained for the developed procedure towardthe determination of PG/HQ were reproducible, because there wasno significant difference between the RSD values of the both ana-lytes. The results are shown in Table 1B. The long term stability ofthe HRP-SiSG/AgNPs/poly(l-Arg)/CPE biosensor was investigatedunder the storage conditions (in a refrigerator, i.e. 4 ◦C); it wasobserved that the activity of immobilized HRP was stable up toone month.

Table 1BThe various parameters determined for PG and HQ.

Parameters PG HQ

Response time (min) 1 1Linear range 0.8 × 10−5 to 30 × 10−5 M 1–150 �MIntercept of calibration curve

(×10−6 A)1.65 0.7056

Slope of calibration curve(×10−6 A/M l−1)

0.3861 0.42

Correlation coefficient 0.9968 0.9994Standard deviation of calibration

curve0.2988 0.0611

Limit of detection (LOD) 6.2 �M 0.57 �MLimit of quantification (LOQ) 20 �M 1.92 �MRepeatability (%RSD) (n = 3) 2.59 3.52Reproducibility (%RSD) (n = 3) 3.14 4.19

n, number of assays.

P. Raghu et al. / Enzyme and Microbial Technology 52 (2013) 377– 385 383

Table 1CRecovery results for PG and HQ in spiked samples.

S. No. Sample matrix Added (�M) Founda (�M) Recovery (%) S.D. (%) Bias

PG HQ PG HQ PG HQ PG HQ PG HQ

1. Mineralized water20 20 20.5 19 102 95 0.18 0.12 +2 −540 40 39 41.5 97.5 103 0.21 0.15 −2.5 +3.760 60 61 59 101.6 98 0.17 0.16 +1 −2

2. Tap water20 40 21 40.5 105 101 0.16 0.13 +5 +140 60 38 58.5 95 97.5 0.19 0.15 −5 −2.560 80 64 81 106 101 0.18 0.14 +6 +1

a Average of four determinations.

Table 1DComparison of different electrochemical/other techniques for the determination of HQ and PG.

Electrode Analyte (linearConc. range)

Technique Limit of detection(LOD)

Refs.

GCEa HQ (3.9–1360 �M)PG (66–440 �M)

CVm

CV3.9 �M33.2 �M

[50]

CNTb-GCE HQ (2.9–1430 �M)PG (66–1660 �M)

CVCV

2.9 �M20.0 �M

HRPc-SiSGd/CPE HQ (5–1000 �M) DPVn 1.5 �M [29]SiO2/Ce HQ (39–1250 �M) DPV 1.6 mmol/l [51]GCE HQ (0.5–30 mg/l) AdSVo 50 mg/l [52]OMIM-PF6/IL-CPEf HQ

(0.01–10 mmol/l)CV 0.81 mmol/l [53]

p-(Glu)CPEg HQ (5–80 �M) DPV 1.5 �M [54]SPCEh PG (10–1000 �M) FIAp 0.33 �M [55]

PG (75–1000 �M) LTCC-microchipsq 75 �M [56]Aui/CNT/PPYj/HRP PG (1.6–22.4 �M) CV 1.24 �M [57]HRP-SiSG/AgNPsk/poly(l-Arg)l/CPE HQ (1–150 �M)

PG (8–300 �M)DPVDPV

0.57 �M6.2 �M

Present work

a GCE, glassy carbon electrode.b CNT, carbon nano tubes.c HRP, horseradish peroxide.d SiSG, silica sol–gel.e SiO2/C, silica on carbon electrode.f OMIM-PF6/IL-CPE-1, octyl-3-methylimidazolium-hexafluorophosphate/ionic liquid-carbon paste electrode.g p-(Glu)CPE, poly(Glutamic acid) carbon paste electrode.h SPCE, screen printed carbon electrodes.i Au, gold electrode.j PPY, poly pyrrole.k AgNPs, silver nanoparticles.l Poly(l-Arg), poly(l-arginine).

m CV, cyclic voltammetry.n DPV, differential pulse voltammetry.o AdSV, adsorptive stripping voltammetry.p FIA, flow injection amperometry.q LTCC-microchips, fabricated by low temperature co-fired ceramic technique.

3.9. Analytical applications

The practical usage of the biosensor was assessed under opti-mized conditions by the determination of PG, HQ in the localtap water and mineralized water samples. The spike and recov-ery experiments were performed through the DPV responses. Theamounts of PG/HQ in the tap water/mineralized water sampleswere determined by calibration method and are summarized inTable 1C. The recoveries were 95–103% and 95–106% for PG and HQrespectively, which clearly indicates the applicability and reliabilityof the proposed method. The detection limit of various electroan-alytical/other methods proposed for the determination of HQ andPG was compared with the present method and shown in Table 1D.

4. Conclusions

In this work, we have successfully developed a methodfor the determination of PG/HQ by using CV and DPV atHRP-SiSG/AgNPs/poly(l-Arg)/CPE. The method of fabricating thebiosensor has many advantages such as ease of fabrication,enhanced electrocatalysis, and long term efficiency in preserving

the activity of enzyme molecules. The biosensor exhibited a fastresponse, a broad liner range, a low detection limit with satisfac-tory stability, repeatability and good potential application in thedetermination of PG/HQ in real samples. Attending to these results,the poly(l-Arg)/AgNPs/SiSG matrix was not only a useful tool forfacilitating the communication between enzyme and electrode, butalso it provides a proper microenvironment for enzyme–substrateinteraction.

Acknowledgement

The authors are gratefully acknowledging the financial supportfrom University Grants Commission (UGC), New Delhi, India, in theform of research project no. F. No. 39-709/2010 (SR).

References

[1] Cui H, He CX, Zhao GW. Determination of polyphenols by high-performanceliquid chromatography with inhibited chemiluminescence detection. Journalof Chromatography A 1999;855:171–9.

384 P. Raghu et al. / Enzyme and Microbial Technology 52 (2013) 377– 385

[2] Asan A, Isildak I. Determination of major phenolic compounds in water byreversed phase liquid chromatography after pre-column derivatization withbenzoyl chloride. Journal of Chromatography A 2003;988:145–9.

[3] Pistonesi MF, Nezio MSD, Centurion ME, Palomeque ME, Lista AG, BandBSF. Determination of phenol, resorcinol and hydroquinone in air sam-ples by synchronous fluorescence using partial least-squares (PLS). Talanta2006;69:1265–8.

[4] Cui H, Zhang QL, Myint A, Ge XW, Liu LJ. Chemiluminescence ofcerium(IV)–rhodamine 6G-phenolic compound system. Journal of Photochem-istry and Photobiology A: Chemistry 2006;181:238–45.

[5] Li SF, Li XZ, Xu J, Wei XW. Flow-injection chemiluminescence determina-tion of polyphenols using luminol–NaIO4–gold nanoparticles system. Talanta2008;75:32–7.

[6] Nagaraja P, Vasantaha RA, Sunitha KR. A sensitive and selective spectro-photometric estimation of catechol derivatives in pharmaceutical preparations.Talanta 2008;55:1039–46.

[7] Moldoveanu SC, Kiser M. Gas chromatography/mass spectrometry versus liquidchromatography/fluorescence detection in the analysis of phenols in main-stream cigarette smoke. Journal of Chromatography A 2007;1141:90–7.

[8] Garcia-Mesa JA, Mateos R. Direct automatic determination of bitternessand total phenolic compounds in virgin olive oil using a pH-based flow-injection analysis system. Journal of Agricultural and Food Chemistry 2007;55:3863–8.

[9] Bai J, Guo LP, Ndamanisha JC, Qi B. Electrochemical properties and simulta-neous determination of dihydroxybenzene isomers at ordered mesoporouscarbon-modified electrode. Journal of Applied Electrochemistry 2009;39:2497–503.

[10] Yu JJ, Du W, Zhao FQ, Zeng BZ. High sensitive simultaneous determination ofcatechol and hydroquinone at mesoporous carbon CMK-3 electrode in compar-ison with multi-walled carbon nanotubes and Vulcan XC-72 carbon electrodes.Electrochimica Acta 2009;54:984–8.

[11] Wang B, Zhang JJ, Pan ZY, Tao XQ, Wang HS. A novel hydrogen peroxidesensor based on the direct electron transfer of horseradish peroxidase immo-bilized on silica–hydroxyapatite hybrid film. Biosensors and Bioelectronics2009;24:1141–5.

[12] Zhang WJ, Li GX. Third-generation biosensors based on the direct electrontransfer of proteins. Analytical Sciences 2004;20:603–9.

[13] Marcus RA. Electron transfer reactions in chemistry: theory and experiment.Angewandte Chemie International Edition 1993;32:1111.

[14] Pingarron JM, Yanez Sedeno P, Gonzalez-cortes A. Gold nanoparticle-basedelectrochemical biosensors. Electrochimica Acta 2008;53:5848–66.

[15] Kafi AKM, Ahmadalinezhad A, Wang JP, Thomas DF, Chen AC. Direct growth ofnanoporous Au and its application in electrochemical biosensing. Biosensorsand Bioelectronics 2010;25:2458–63.

[16] Yin H, Ai S, Shi W, Zhu L. A novel hydrogen peroxide biosensor basedon horseradish peroxidase immobilized on gold nanoparticles–silk fibroinmodified glassy carbon electrode and direct electrochemistry of horseradishperoxidase. Sensors and Actuators B 2009;137:747–53.

[17] Xu J, Shang F, Luong JHT, Razeeb KM, Glennon JD. Direct electrochemistry ofhorseradish peroxidase immobilized on a monolayer modified nanowire arrayelectrode. Biosensors and Bioelectronics 2010;25:1313–8.

[18] Zhu Z, Li X, Wang Y, Zeng Y, Sun W, Huang X. Direct electrochemistryand electrocatalysis of horseradish peroxidase with hyaluronic acid-ionicliquid–cadmium sulfide nanorod composite material. Analytica Chimica Acta2010;670:51–6.

[19] Yang Y, Yang G, Huang Y, Bai H, Lu X. A new hydrogen peroxide biosensor basedon gold nanoelectrode ensembles/multiwalled carbon nanotubes/chitosanfilm-modified electrode. Colloids and Surfaces A 2009;340:50–5.

[20] Wang S, Xie F, Liu G. Direct electrochemistry and electrocatalysis ofheme proteins on SWCNTs-CTAB modified electrodes. Talanta 2009;77:1343–50.

[21] Cao DF, He PL, Hu NF. Electrochemical biosensors utilizing electrontransfer in heme proteins immobilised on Fe3O4 nanoparticles. Analyst2003;128:1268–74.

[22] Saroj K, Pradip N. Sunlight-induced covalent immobilization of proteins.Talanta 2007;71:1438–40.

[23] Qu S, Huang F, Chen G, Yu SN, Kong JL. Magnetic assembled electrochemicalplatform using Fe2O3 filled carbon nanotubes and enzyme. ElectrochemistryCommunications 2007;9:2812–6.

[24] Wang JW, Qu M, Di JW, Gao YS, Wu Y, Tu YF. A carbon nanotube/silicasol–gel architecture for immobilization of horseradish peroxidase for elec-trochemical biosensor. Bioprocess and Biosystems Engineering 2007;30:289–96.

[25] Morrin A, Wilbeer F, Ngamna O, Moulton SE, Killard AJ, Wallace GG, et al. Novelbiosensor fabrication methodology based on processable conducting polyani-line nanoparticles. Electrochemistry Communications 2005;7:317–22.

[26] Shao Y, Jin YD, Wang JL, Wang L, Zhao F, Dong SJ. Conducting polymerpolypyrrole supported bilayer lipid membranes. Biosensors and Bioelectronics2005;20:1373–9.

[27] Jin W, Brennan JD. Properties and applications of proteins encapsulated withinsol–gel derived materials. Analytica Chimica Acta 2002;461:1–36.

[28] Aurobind SV, Amirthalingam KP, Gomathi H. Sol–gel based surface modificationof electrodes for electro analysis. Advances in Colloid and Interface Science2006;121:1–7.

[29] Reddaiah K, Madhusudana Reddy T, Raghu P, Gopal P. Sol–gel immobi-lized horseradish peroxidase modified carbon paste electrode towards the

determination of hydroquinone in non-aqueous solvents: a voltammetricstudy. Analytical & Bioanalytical Electrochemistry 2012;4:372–85.

[30] Jaidev LR, Narasimha G. Fungal mediated biosynthesis of silver nanopar-ticles, characterization and antimicrobial activity. Colloids and Surfaces B2010;81:430–3.

[31] Raghu P, Kumara Swamy BE, Madhusudana Reddy T, Chandrashekar BN,Reddaiah K. Sol–gel immobilized biosensor for the detection of organophos-phorous pesticides: a voltammetric method. Bioelectrochemistry 2012;83:19–24.

[32] Raghu P, Madhusudana Reddy T, Kumara Swamy BE, Chandrashekar BN,Reddaiah K, Sreedhar M. Development of AChE biosensor for the determi-nation of methyl parathion and monocrotophos in water and fruit samples:a cyclic voltammetric study. Journal of Electroanalytical Chemistry 2012;665:76–82.

[33] Anitha K, Venkata Mohan S, Jayarama Reddy S. Development of acetyl-cholinesterase silica sol–gel immobilized biosensor – an application towardsoxydemeton methyl detection. Biosensors and Bioelectronics 2004;20:848–56.

[34] Hong Y, Yuanyuan S, Xinhua L, Yuhai T, Liying H. Electrochemical character-ization of poly(eriochrome black T) modified glassy carbon electrode and itsapplication to simultaneous determination of dopamine, ascorbic acid and uricacid. Electrochimica Acta 2007;52:6165–71.

[35] Zhang Y, He P, Hu N. Horseradish peroxidase immobilized in TiO2 nanoparticlefilms on pyrolytic graphite electrodes: direct electrochemistry and bioelectro-catalysis. Electrochimica Acta 2004;49:1981–8.

[36] Laviron E. The use of linear potential sweep voltammetry and of a.c. voltam-metry for the study of the surface electrochemical reaction of strongly adsorbedsystems and of redox modified electrodes. Journal of Electroanalytical Chem-istry 1979;100:263–70.

[37] Li J, Dong S. The electrochemical study of oxidation-reduction prop-erties of horseradish peroxidase. Journal of Electroanalytical Chemistry1997;431:19–22.

[38] Zheng Y, Qin LX. Modified electrode based on immobilizing horseradish perox-idase on nano-gold with choline covalently modified glassy carbon electrodeas a base. Chinese Journal of Analytical Chemistry 2008;36:604–8.

[39] Liu X, Huang Y, Shang L, Wang X, Xiao H, Li G. Electron transfer reactivity and thecatalytic activity of horseradish peroxidase incorporated in dipalmitoylphos-phatidic acid films. Bioelectrochemistry 2006;68:98–104.

[40] Liu S, Ju H. Renewable reagentless hydrogen peroxide sensor based on directelectron transfer of horseradish peroxidase immobilized on colloidal gold-modified electrode. Analytical Biochemistry 2002;307:110–6.

[41] Delvaux M, Walcarius A, Demoustier-Champagne S. Electrocatalytic H2O2

amperometric detection using gold nanotube electrode ensembles. AnalyticaChimica Acta 2004;525:221–30.

[42] Wang BQ, Li B, Deng Q, Dong SJ. Amperometric glucose biosensorbased on sol–gel organic–inorganic hybrid material. Analytical Chemistry1998;70:3170–4.

[43] Madhusudana Reddy T, Reddy SJ. Differential pulse adsorptive strippingvoltammetric determination of nifedipine and nimodipine in pharmaceuticalformulations, urine, and serum samples by using a clay-modified carbon-pasteelectrode. Analytical Letters 2004;37:2079–98.

[44] Madhusudana Reddy T, Sreedhar M, Reddy SJ. Voltammetric behavior ofCefixime and Cefpodoxime Proxetil and determination in pharmaceuticalformulations and urine. Journal of Pharmaceutical and Biomedical Analysis2003;31:811–8.

[45] Camacho C, Matías JC, Chico B, Cao R, Gómez L, Simpson BK, et al. Ampero-metric biosensor for hydrogen peroxide, using supramolecularly immobilizedhorseradish peroxidase on the �-cyclodextrin-coated gold electrode. Electro-analysis 2007;19:2538–42.

[46] Di J, Shen C, Peng S, Tu X, Li S. A one-step method to construct a third-generationbiosensor based on horseradish peroxidase and gold nanoparticles embeddedin silica sol–gel network on gold modified electrode. Analytica Chimica Acta2005;553:196–200.

[47] Xu X, Liu S, Ju H. A novel hydrogen peroxide sensor via the direct electro-chemistry of horseradish peroxidase immobilized on colloidal gold modifiedscreen-printed electrode. Sensors 2003;3:350–60.

[48] Xu JZ, Zhu JJ, Wu Q, Hu Z, Chen HY. An amperometric biosensor based on thecoimmobilization of horseradish peroxidase and methylene blue on a carbonnanotubes modified electrode. Electroanalysis 2003;15:219–24.

[49] Chut SL, Li J, Tan SN. Reagentless amperometric determination of hydrogenperoxide by silica sol–gel modified biosensor. Analyst 1997;122:1431–4.

[50] Ziyatdinova G, Gainetdinova A, Morozov M, Budnikov H, Grazhulene S, Red’kinA. Voltammetric detection of synthetic water-soluble phenolic antioxidantsusing carbon nanotube based electrodes. Journal of Solid State Electrochemistry2012;16:127–34.

[51] Canevari TC, Arenas LT, Landers R, Roxgério C, Gushikema Y. Simultaneouselectroanalytical determination of hydroquinone and catechol in the presenceof resorcinol at an SiO2/C electrode spin-coated with a thin film of Nb2O5.Analyst 2013;138:315–24.

[52] Niaz A, Sirajuddin Shah A, Mahesar SA, Rauf A. Adsorptive stripping voltam-metric determination of hydroquinone using an electrochemically pretreatedglassy carbon electrode. Pakistan Journal of Analytical & Environmental Chem-istry 2008;9:110–7.

[53] She Y, Tang Y, Liu H, He P. Electrochemical determination of hydroquinoneusing hydrophobic ionic liquid-type carbon paste electrodes. Chemistry CentralJournal 2010;4:17.

P. Raghu et al. / Enzyme and Microbial Technology 52 (2013) 377– 385 385

[54] Wang L, Huang P, Bai J, Wang H, Zhang L, Zhao Y. Direct simultaneous elec-trochemical determination of hydroquinone and catechol at a poly(glutamicacid) modified glassy carbon electrode. International Journal of ElectrochemicalScience 2007;2:123–32.

[55] Feng P-S, Wang S-M, Su W-Y, Cheng S-H. Electrochemical oxidation, sensitivedetermination of Pyrogallol at Preanodized screen-printed carbon electrodes.Journal of the Chinese Chemical Society 2011;59:1–8.

[56] Goldbach M, Axthelm H, Keusgen M. LTCC-based microchips for the elec-trochemical detection of phenolic compounds. Sensors and Actuators B2006;120:346–51.

[57] Korkut S, Keskinler B, Erhan E. An amperometric biosensor based onmultiwalled carbon nanotube-poly(pyrrole)-horseradish peroxidase nanobio-composite film for determination of phenol derivatives. Talanta 2008;76:1147–52.

Sol–gel immobilized biosensor for the detection of organophosphorous pesticides:A voltammetric method

P. Raghu a, B.E. Kumara Swamy b,⁎, T. Madhusudana Reddy a,⁎, B.N. Chandrashekar b, K. Reddaiah a

a Department of Chemistry, S.V.U. College of Sciences, Sri Venkateswara University, Tirupati-517502, Andhra Pradesh, Indiab Department of P.G. Studies and Research in Industrial Chemistry, Kuvempu University, Shankaraghatta-577 451, Shimoga, Karnataka, India

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

Article history:Received 20 June 2011Received in revised form 1 August 2011Accepted 1 August 2011Available online 11 August 2011

Keywords:AcetylcholinesteraseAcetylthiocholine chlorideCarbon paste electrodeImmobilizationMethyl parathion and acephate

Organophosphorous compounds are important neuroactive molecules whose presence exhibits significantanalytical challenges. An acetylcholinesterase (AChE) based amperometric biosensor was developed by silicasol–gel film immobilization of the enzyme onto the carbon paste electrode. The mono enzyme biosensor wasused for the determination of two organophosphorous compounds such as methyl parathion (MP) andacephate in 0.1 M phosphate buffer (pH 7.0). The substrate used was acetylthiocholine chloride (ASChCl)confirmed the formation of thiocholine and it was electrochemically oxidized giving significant increase inanodic peak current around at 0.60 V versus calomel electrode. The influence of pH, enzyme loading andsubstrate concentration on the response of the biosensor was investigated. The monoenzyme biosensorprovided linearity to methyl parathion and acephate in the concentration range of 0.1–0.5 ppb and 50–750 ppb with an incubation time of 20 min and 4 min. The detection limits under the optimum workingconditions were found to be 0.08 ppb for methyl parathion and 87 ppb for acephate. The sensor shows goodoperational stability 89% of its original activity for 60 successive measurements.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Organophosphorous compounds are considered to be the mosttoxic. They are used as pesticides, insecticides and chemical waragents. The high toxicity of organophosphorous neurotoxics and theirlarge use in modern agriculture practices has increased publicconcerns, health risks and the consequent contamination of water,food sources [1]. The use of any technology for detoxification oforganophosphorous compounds, performed in laboratories, will needthe development of analytical tools of high performance in order tocontrol the concentration of neurotoxics.

The techniques such as gas chromatography, liquid chromatogra-phy and thin film chromatography couple with different detectors andthe different types of spectroscopy are the most commonly usedmethods. However, these techniques are time consuming, expensiveand demand a qualified and experienced staff and cannot be used forcontinuous monitoring. Biological techniques such as immunoassaysand inhibition of cholinesterase activity by colorimetric techniquesare also used for the determination of organophosphorous com-pounds. Immunoassays require long analysis time and pre-sampletreatment which are expensive enough and these techniques are notsuitable for continuous monitoring [2].

Biosensors are sensitive and can be used for pesticide determina-tion. In these sensors, the inhibition of AChE can be measured byelectrochemical techniques such as pH-shift potentiometry [3]. Themain disadvantage of this technique is its strict requirement for lowbuffer capacity of analyte solution. This may lead to significantcomplexities and uncertainness in the measurement of the organo-phosphorous compounds. The sensitivity of pH-based analyticaltechniques is less than that of amperometric methods.

Many immobilization methods have been employed to fabricatebiosensors with high enzyme activity and fast electro transfer rates[4]. Sol–el immobilization platforms can be preferred to otherencapsulation, entrapment of sensing agents with in a polymericmatrix, since some of these procedures are tedious and results in poorstability and perturbed function, require expensive reagents. There-fore many sol–gel derived enzyme biosensors have been developed atthe research level to monitor glucose, lactate, cholesterol, dopamine,H2O2, phenols and urea [5–11]. Recent development in the area ofamperometric biosensors with sol–gel encapsulation of enzymespecies as an immobilization matrix is very encouraging and offerspotential advantages. These advantages include the ability of sol–gel(i) to form at low temperatures and under chemical, mechanicalstability and offers negligible swelling, (ii) open to a wide variety ofchemical modifications based on the inclusion of various polymeradditives, redox modifiers and organically modified silanes, resultingin electrically conducting materials, (iii) to exhibit tunable pore sizeand pore distribution, which allows small molecules and ions todiffuse into the matrix while larger biomolecules remain trapped in

Bioelectrochemistry 83 (2012) 19–24

⁎ Corresponding authors.E-mail addresses: kumaraswamy21@yahoo.com (B.E. Kumara Swamy),

tmsreddysvu@gmail.com (T. Madhusudana Reddy).

1567-5394/$ – see front matter © 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.bioelechem.2011.08.002

Contents lists available at SciVerse ScienceDirect

Bioelectrochemistry

j ourna l homepage: www.e lsev ie r.com/ locate /b ioe lechem

the pores, simplicity of preparation without any kinds of modifica-tions [12].

This work describes the construction of a simple voltammetricmonoenzyme biosensor for the direct, sensitive and selective determi-nation of methyl parathion and acephate using acetylcholinesterase bysol–gel immobilization on the surface of carbon paste electrode. Thestructural and molecular formulae of methyl parathion and acephatewere shown in Table 1. This paper assesses experimental parameterssuch as pH, scan rate and enzyme loading have been investigated toevaluate the conditions for the best performance of the biosensor.

2. Experimental

2.1. Materials

All chemicals were obtained from commercial sources and usedwithout further purification. Acetylcholinesterase (EC: 3.1.1.7, type VI-S/1.5 mg, electric eel source, 500U/1.5 mg), acetylthiocholine chloridewere purchased from Sigma-Aldrich chemicals, USA. Tetraethylorthosi-licate (TEOS), cetyl trimethyl ammoniumbromide (CTAB), Triton-X-100were obtained from Sigma-Aldrich chemicals co. USA. Methyl parathionandacephatewere obtained fromaccustandard solutions company,USA.The pesticide stock solution was prepared dissolving in acetone (GRgrade) solution. The graphite fine powder was procured from Lobochemie and silicon oil (Himedia) and acetone (GR grade) was obtainedfromMerck specialities Pvt.Ltd. Phosphate buffer solution was preparedbymixing theappropriatequantity of 0.1 Maqueous sodiumdihydrogenphosphate monohydrate and 0.1 M aqueous disodium hydrogenphosphate. All the chemicals were of analytical grade and aqueoussolutions were prepared with double distilled water. The enzyme stocksolution was stored at −20 °C. All stock and working solutions ofchemicals were stored at 4 °C.

2.2. Apparatus

The electrochemical experiments were carried out using a modelCH-660C (CH-Instruments). All the experiments were carried out in aconventional three electrode electrochemical cell. The electrode systemcontained a working electrode was sol–gel immobilized enzymemodified carbon paste electrode, a platinumwire as a counter electrodeand saturated calomel electrode as reference electrode. All theexperiments were carried out at room temperature 25±2 °C.

2.3. Preparation of sol–gel immobilized enzyme modified carbon pasteelectrode

The carbon paste electrode was prepared as follows; 70% graphitepowder and 30% silicon oil were mixed by hand to produce ahomogenous a homemade carbon paste electrode and smoothed onweighing paper. A homogenous TEOS silica sol–gel was prepared bymixing 2 ml of TEOS, 1 ml of H2O, 50 μl of 0.1 MHCl, 25 μl of 10% Triton-

X-100. Themixturewas stirred for 1 hr until a clear sol was formed. Thesol can be stored for several months when refrigerated at −20 °C.

The 5 μl of stock sol–gel solution was vortexed with 45 μl ofphosphate buffer containing 0.5 U of enzyme stock solution. The 3 μlof the enzyme sol was spread on the electrode surface. This film wasallowed to polymerize at room temperature for 3–5 min. Theelectrode was gently washed with phosphate buffer (pH 7.0) andwas used for further experimental procedure [13]. The 0.03U ofenzyme was immobilized on the electrode surface.

3. Results and discussion

3.1. Electrochemistry of acetylthiocholine chloride (ASChCl) at thesurface of AChE enzyme immobilized graphite paste electrode

The electrochemical behavior of thiocholine which was releasedfrom enzymatic hydrolysis of acetylthiocholine chloride in presence ofAChE. Fig. 1 shows the cyclic voltammograms of the sensor in thepresence and absence of 1 mM substrate in 0.1 M phosphate buffer(pH 7.0) at a scan rate of 10 mVs−1. In the absence of substrate, theenzyme electrode gave no response and only a small backgroundcurrent was observed [dashed line]. When the substrate was added tothe buffer solution, a relatively larger anodic current at potentials of0.65 V was observed. In the present electrochemical approach, theelectrode response was proportional to the oxidation of an electro-active species produced by the enzymatic reaction. The mechanism ofthe reaction has been shown in Scheme (1)[14].

The increased concentrations of substrate showed gradual increasein peak current response and finally saturated at which current valuesobserved were limited. The calibration plot of the typical enzyme–substrate reaction was shown in Fig. 2. The kinetics of theimmobilized enzyme showing diphasic in nature, i.e. at low amountsof substrate, the enzyme rate was varied with concentration (firstorder ) and at high concentrations of substrate it reach plateau levelthe rate was independent of concentration ( zero order ). Theapparent Michaelis–Menten constant was (Km

app) 450 μM which wascalculated from the Lineweaver–Burk equation. Analysis of the slopeand intercept for the plot of reciprocal of current response (1/V)versus reciprocal of substrate concentration (1/S) allowed thedetermination of Km

app [13].

3.2. The effect of scan rate and pH

The effect of scan rate for acetylthiocholine chloride (ASChCl) wasstudied at enzyme modified carbon paste electrode. It showedincrease in the oxidation peak current with increase in scan ratefrom 5 to 50 mVs−1. The graph of current (Ipa) versus square root ofscan rate (√ν) was plotted. The graph obtained resulted with goodlinearity Fig. 3. The correlation coefficient was 0.9384, which indicatethe electrode reaction was diffusion controlled process [15–17].

Table.1The structural and molecular formulae of methyl parathion and acephate.

Pesticide Molecular formula Structure

Methyl parathion C8H10O5NPS

Acephate C4H10NO3PSFig. 1. Cyclic voltammogram of sol–gel immobilized acetylcholinesterase electrode in0.1 M phosphate buffer, pH 7.0 and 0.1 M KCl (a) without substrate (dashed line),(b) with 1 mM substrate (solid line).

20 P. Raghu et al. / Bioelectrochemistry 83 (2012) 19–24

The effect of pH on the electrochemical response of the enzymeelectrode towards the 1 mM substrate over the pH range 5.5 to 9.0 in0.1 M phosphate buffer solution was illustrated in Fig. 4[A]. Theresulting profile showed maximum sensitivity of the enzymeelectrode at pH 7.0. The immobilized acetylcholinesterase enzymeelectrode showed an optimum pH range of 7.0–8.0 [18]. The potentialdiagram was constructed by plotting the graph of anodic peakpotential Epa Vs pH of the solution and shown in Fig. 4[B]. The pHdependence of oxidation peak potential of substrate, Ep=0.9657+0.052 pH (r2=0.9905). The graph has good linearity with a slope of52 mV/pH, this behavior was nearly obeyed the Nernst equation forequal number of electron and proton transfer reaction [19,20].

3.3. Effect of enzyme concentration

Fig. 5 shows the effect of enzyme concentration which was used infabrication of monoenzyme biosensor based on its response. It can befollowed from the obtained data that the enzyme loading from 0.03 to0.3U into the sol–gel matrix produced increased response with 1 mMsubstrate and resulted in the increased sensitivity and shorter responsetimes. The rate of enzyme catalyzed reaction was dependent on theamount of enzyme immobilized. The lowest amount of enzyme wasnecessary to achieve the lowest detection limit of pesticides [21–24].

3.4. Measurement of organophosphorous pesticides

AChE sensors have been used to carry out inhibition studies withthe two pesticides: methyl parathion and acephate. Sol–gel immobi-lization plot form used could provide wider concentration range ofpesticide detection sensitive to ppb level. The detection was based on

Scheme.1. The mechanism of the enzyme catalyzed reaction.

Fig. 2. Biosensor response for increasing concentration of acetylthiocholine chloride in0.1 M phosphate buffer containing 0.1 M KCl, pH 7.0.

Fig. 3. The effect of square root of scan rate on anodic peak current for 1 mM ASChCl in0.1 M phosphate buffer, pH 7.0 and 0.1 M KCl.

Fig. 4. [A] Effect of pH on the enzyme electrode response. [B] Graph of different pH vsEpa to 1 mM ASChCl.

Fig. 5. Biosensor response for increasing concentration of enzyme on the surface of theelectrode in 0.1 M phosphate buffer containing 0.1 M KCl, pH 7.0.

21P. Raghu et al. / Bioelectrochemistry 83 (2012) 19–24

the measurement of current response of the substrate 1 mM ASChClby placing the biosensor in a buffered solution (0.1 M phosphatebuffer and 0.1 M KCl at pH 7.0). The response of the biosensor wasmeasured as the current increases in relation with the base currentwhen the substrate was added to the buffer (Ii). After that, thebiosensor was placed for 1 min in contact with the sample containingthe pesticides, which inhibit the AChE and the same measurementwas repeated (IF). The percentage of inhibition and residual enzymeactivity were calculated as follow: [25]

Inhibition % I%ð Þ= Ii–IFð Þ= Ii½ � × 100Residual enzyme activity % REA %ð Þ= IF = Ii½ � × 100

Methyl parathion and acephate pesticides are known to inhibit thereaction between AChE and acetylthiocholine chloride, hence theyhave been used widely as toxic reference to test acetylcholinesterasebiosensors.

Quantitative analysis of individual pesticides was carried outaccording to the above procedure. Fig. 6 shows the DPV voltammogramfor substrate alone and pesticide solution where complete inhibitionoccurred. Calibration plots based on the dependence of the % inhibitionon concentration were linear and shown in Fig. 7[A] and [B] for methylparathion and acephate respectively. The detection limit and limit ofquantification values were 0.08 ppb, 87 ppb and 0.28 ppb, 292 ppb formethyl parathion and acephate respectively.When compared the abovetwo pesticides methyl parathionwasmore toxic than acephate becauseit has less DL value than acephate. Thus, these results clearly indicatethat the proposed electrochemical method of analysis was reliable forthe determination of individual pesticides. The calibration plot of %residual enzyme activitywith incubation time formethyl parathion andacephatewas shown in Fig. 8[A] and [B]. When the concentration ofpesticide increases the residual enzyme activity of the enzymedecreases with time. Methyl parathion and acephate was shown inFig. 9[A] and [B]. Determination of DL and QL were carried by using thefollowing expression [15,26–29].

DL = 3Sb=SQL = 10Sb=S

Where Sb is standard deviation, S is the slope of the working curve,DL is the detection limit, QL is the quantification limit. Table 2 shows thevarious parameters determined for methyl parathion and acephate.

Fig. 6. Differential pulse voltammogramof (a) substrate alone, (b)with pesticide solution.

Fig. 7. Inhibition plots of [A] methyl parathion after 20 min incubation time, [B] acephateafter 4 min incubation time with the measurement conditions in 100 mM phosphatebuffer/KCl.

Fig. 8. The effect of incubation time at various inhibitor concentrations on the activity ofimmobilized enzyme in 0.1 M phosphate buffer/KCl, pH 7.0. [A] For methyl parathion:(a) 0.1 ppb, (b) 0.2 ppb, (c) 0.3 ppb, (d) 0.4 ppb, (e) 0.5 ppb. [B] For acephate: (a) 50 ppb,(b) 100 ppb, (c) 200 ppb, (d) 350 ppb, (e) 500 ppb, (f) 700 ppb, (g) 750 ppb.

22 P. Raghu et al. / Bioelectrochemistry 83 (2012) 19–24

The robustness of the developedmethod was evaluated by studyingthe concept of repeatability (new electrode, same standard solution,sameday, same analyst, n is number of assays) and reproducibility (newelectrode, new standard solution, different days, different analyst, n isnumber of assays) of the biosensor towards the inhibition of pesticides[26,27]. To study this experiment the chosen concentration of the stocksolutions of methyl parathion and acephate were 0.4 ppb and 600 ppb.The results obtained for thedevelopedprocedure towards the inhibitionof both the pesticides was reproducible, because there was nosignificant difference between the RSD values of the both the pesticides.The results were shown in Table 2. The long term stability of the AChEbiosensorwas investigated under the storage conditions (at 4°C), it wasnoticed that the activity of immobilized AChEwas stable up to 1 month.

3.5. Biosensor response characteristics

Fig. 10 shows the operational stability of the biosensor was alsoconfirmed by examining its repeated response to 1 mM ASChCl for1 hr. The enzyme electrode shows good operational stability retaining89% of its original activity with 60 successive measurements withsubstrate alone. The activity of the entrapped acetylcholinesterase inthe sol–gel film was stable and there was no decrease up to 50measurements in the absence of inhibitor. The enzyme electrode inthe presence of inhibitor could not retain its activity instead droppedto 34% of its original value after 20 measurements.

4. Conclusions

The research described in this paper resulted in voltammetricmethod for the determination of organophosphorous pesticides.Biosensor prepared by using simple sol–gel technology to immobilizethe acetylcholinesterase takes minimal preparation time. The detec-tion limit of the AChE sensor was directly related to the capacity of thepesticide to inhibit AChE. The new sensors are of single use and can beproduced at low cost. The AChE biosensors possess distinct advan-tages, including monitoring of hydrophobic substrates and relativeease of enzyme immobilization.

Acknowledgements

The authors are very much thankful to the Department of Scienceand Technology Government of India, NewDelhi for the funding giventhrough project no. SR/FT/CS-025/2009.

References

[1] H. Tavakoli, H. Ghourchian, Mono-enzyme biosensor for the detection oforganophosphorous compounds, J. Iran. Chem. Soc. 7 (2010) 322–332.

[2] Nicole Jaffrezic-Renault, New trends in biosensors for organophosphoruspesticides, Sensors 1 (2001) 60–74.

[3] F. Arduini, F. Ricci, C.S. Tauta, D. Moscone, A. Amine, G. Palleschi, Detection ofcarbamic and organophosphorous pesticides in water samples using a cholines-terase biosensor based on Prussian Blue-modified screen-printed electrode, Anal.Chim. Acta 580 (2006) 155–162.

[4] Valber A. Pedrosa, Josiame ca etano, Sergio A.S. Machado, Renato S. Freire, MauroBertotti, Acetylcholinesterase immobilization on 3-mercaptopropionic acid selfassembled monolayer for determination of pesticides, Electroanalysis 19 (2007)1415–1420.

[5] S. Sampth, O. Lev, Anal. Chem. 68 (1996) 2015–2021.[6] T.M. Park, E.I. Lcouoha, M.R. Smyth, R. Freoney, A.J. Mcshane, Sol–gel based

amperometric biosensor incorporating an osmium redox polymer as mediator fordetection of L-lactate, Talanta 44 (1997) 973–978.

[7] T. Yao, K. Takashima, Amperometric biosensorwith a compositemembraneof sol–gelderived enzyme film and electrochemically generated poly(1,2-diaminobenzene)film, Biosens. Bioelectron. 13 (1998) 67–73.

Fig. 9. The variation of residual enzyme activity with different inhibitor concentrationswith time in 0.1 M phosphate buffer/KCl, pH 7.0. [A] For methyl parathion, [B] foracephate.

Table.2The various parameters determined for methyl parathion and acephate.

Parameters Methyl parathion Acephate

Response time (min) 1 1Incubation time (min) 20 4Linear range (ppb) 0.1 – 0.5 50 – 750Intercept of calibration curve (10-7A) 4.2 37.66Slope of calibration curve (10-7A / μgL-1) 196 0.08Correlation coefficient 0.9884 0.9955Standard deviation 5.4894 2.3418Detection limit (DL) (ppb) 0.08 87Quantification limit (QL) (ppb) 0.28 292Repeatability (%RSD) (n=3) 2.85 3.27Reproducibility (%RSD) (n=3) 3.73 4.09

n=number of assays.

Fig. 10. Dependence of residual activity of the enzyme in the presence of 1 mM substratewith successive number of measurements: (a) in the absence, (ii) in the presence of100 ppb of inhibitor.

23P. Raghu et al. / Bioelectrochemistry 83 (2012) 19–24

[8] P.C. Pandey, S. Upadhyay, I. Tiwari, G. Singh, V.S. Tripathi, A novel ferroceneencapsulated palladium-linked ormosil-based electrocatalytic dopamine biosen-sor, Sens. Actuators, B 75 (2001) 48–55.

[9] T.M. Park, Amperometric determination of hydrogen peroxide by utilizing a sol–gel-derived biosensor incorporating an osmium redox polymer as mediator, Anal.Lett. 32 (1999) 287–298.

[10] J. Li, L.S. china, N.K. Goh, S.N. Tan, Silica sol–gel immobilized amperometricbiosensor for the determination of phenolic compounds, Anal. Chim. Acta 362(1998) 203–211.

[11] P.C. Pandey, G. Singh, Tetraphenylborate doped polyaniline based novel pH sensorand solid-state urea biosensor, Talanta 55 (2001) 773–782.

[12] J. Lin, C.W. Brown, Sol–gel glass as a matrix for chemical and biochemical sensing,Trends Anal. Chem. 16 (1997) 200–211.

[13] K. Anitha, S. VenkataMohan, S. Jayarama Reddy, Development of acetylcholines-terase silica sol–gel immobilized biosensor—an application towards oxydemetonmethyl detection, Biosens. Bioelectron. 20 (2004) 848–856.

[14] G.S. Nunes, D. Barcelo, B.S. Grabaric, J.M. Diazcruz, M.L. Ribeiro, Evaluation of ahighly sensitive amperometric biosensor with low cholinesterase chargeimmobilized on a chemically modified carbon paste electrode for tracedetermination of carbamates in fruit, vegetable and water samples, Anal. Chim.Acta 399 (1999) 37–49.

[15] Umesh Chandra, B.E. Kumara Swamy, Ongera Gilbert, B.S. Sherigara, Voltammetricresolution of dopamine in the presence of ascorbic acid and uric acid at poly(calmagite) film coated carbon paste electrode, Electrochem. Acta 55 (2010)7166–7174.

[16] Ongera Gilbert, B.E. Kumara Swamy, Umesh Chandra, B.S. Sherigara, Simultaneousdetection of dopamine and ascorbic acid using polyglycine modified carbon pasteelectrode: a cyclic voltammetric study, J. Electroanal. Chem. 636 (2009) 80–85.

[17] S. Chithravathi, B.E. Kumara Swamy, Umesh Cahndra, G.P. Mamatha, B.S.Sherigara, Electrocatalytic oxidation of sodium levothyroxine with phenylhydrazine as a mediator at carbon paste electrode: a cyclic voltammetric study,J. Electroanal. Chem. 645 (2010) 10–15.

[18] N. Mionelto, J.-L. Marty, Karube, Acetylcholinesterase in organic solvents for thedetection of pesticides: biosensor application, Biosens. Bioelectron. 9 (1994)463–470.

[19] B.D. Jones, J.D. Ingle Jr., Evaluation of immobilized redox indicators as reversible,in situ redox sensors for determining Fe(III)-reducing conditions in environmen-tal samples, Talanta 55 (2001) 699–714.

[20] M.C. Shen, H.C. Gheng, V.S. Vasantha, Preparation and electrocatalytic propertiesof the TBO/nafion chemically-modified electrodes, J. Electroanal. Chem. 588(2006) 235–243.

[21] E. Suprun, G. Evtugun, H. Budnikov, F. Ricci, D. Moscone, G. Palleschi,Acetylcholinesterase sensor based on screen-printed carbon electrode modifiedwith prussian blue, Anal. Bioanal. Chem. 383 (2005) 597–604.

[22] S. Sotiropoulou, N.A. Chaniotakis, Lowering the detection limit of the acetylcho-linesterase biosensor using a nanoporous carbon matrix, Anal. Chim. Acta. 530(2005) 199–204.

[23] A. Ivanov, G. Evtugyn, H. Budnikov, Riccif, D. Moscone, G. Palleschi, Cholinesterasesensors based on screen-printed electrodes for detection of organophosphorusand carbamic pesticides, Anal. Bioanal. Chem. 377 (2003) 624–631.

[24] G.A. Evtugyn, H.C. Budnikov, E.B. Nikolskaya, Sensitivity and selectivity ofelectrochemical enzyme sensors for inhibitor determination, Talanta 46 (1998)465–484.

[25] Alian Hildebrandt, Jordi Ribas, Ramon Bragos, Jean - Louis Marty, MariusTresanchez, Silvia Lacorte, Development of a portable biosensor for screeningneurotoxic agents in water samples, Talanta 75 (2008) 1208–1213.

[26] T. Madhusudana Reddy, S.J. Reddy, Differential pulse adsorptive strippingvoltammetric determination of nifedipine and nimodipine in pharmaceuticalformulations, urine, and serum samples by using a clay-modified carbon-pasteelectrode, Anal. Lett. 37 (2004) 2079–2098.

[27] T. Madhusudana Reddy, M. Sreedhar, S.J. Reddy, Voltametric behavior of Cefiximeand Cefpodoxime Proxetil and determination in pharmaceutical formulations andurine, J. Pharm. Biomed. Anal. 31 (2003) 811–818.

[28] Djenaine De Souza, Sergio A.S. Machado, Electroanalytical method for determi-nation of the pesticide dichlorvos using gold-disk microelectrodes, Anal. Bioanal.Chem. 382 (2005) 1720–1725.

[29] Rajesh.N. Hegde, B.E. Kumara Swamy,Nagaraj.P. Shetti, Sharanappa.T. Nandibewoor,Electro-oxidation and determination of gabapentin at gold electrode, J. Electroanal.Chem. 635 (2009) 51–57.

24 P. Raghu et al. / Bioelectrochemistry 83 (2012) 19–24

Development of AChE biosensor for the determination of methyl parathion andmonocrotophos in water and fruit samples: A cyclic voltammetric study

P. Raghu a, T. Madhusudana Reddy a,⇑, B.E. Kumara Swamy b,⇑, B.N. Chandrashekar b, K. Reddaiah a,M. Sreedhar c

a Electrochemical Research Laboratory, Department of Chemistry, S.V.U. College of Sciences, Sri Venkateswara University, Tirupati 517 502, Andhra Pradesh, Indiab Department of P.G. Studies and Research in Industrial Chemistry, Kuvempu University, Shankaraghatta 577 451, Shimoga, Karnataka, Indiac Pesticide Residue Analysis Laboratory, Regional Plant Quarantine Station, Meenambakkam, Chennai 600 027, Tamilnadu, India

a r t i c l e i n f o

Article history:Received 6 August 2011Received in revised form 16 November 2011Accepted 18 November 2011Available online 26 November 2011

Keywords:Acetylcholinesterase (AChE)AcetylthiocholinechlorideSilica sol–gelImmobilizationMethyl parathion and monocrotophos

a b s t r a c t

AChE based biosensor for the determination of methyl parathion and monocrotophos directly in the stan-dard solutions and spiked (water and fruit) samples have been evaluated and electrochemically charac-terized by cyclic voltammetric technique. The biosensor was applied for the direct determination ofpesticides in spiked samples at ppb concentration levels without any sample pretreatment steps. The fab-ricated acetyl cholinesterase based biosensor entrapped using silica sol–gel onto the carbon paste elec-trode was used as a working electrode. Acetylcholinesterase catalyses the cleavage of acetylthiocholinechloride (ASChCl or substrate) to thiocholine and it was electrochemically oxidized giving significant ano-dic peak current around at 0.60 V versus saturated calomel electrode. The effect of substrate concentra-tion, enzyme loading and pH on the response of the biosensor was investigated. The electrochemicalexperiments were performed in 0.1 M phosphate buffer solution (pH 7.0) and 0.1 M KCl at room temper-ature. The limit of detection and limit of quantification values were found to be 0.04 ppb, 47 ppb and0.14 ppb, 158 ppb for methyl parathion and monocrotophos, respectively. The analytical curve obtainedfor methyl parathion and monocrotophos in pure solutions showed excellent linearity in the concentra-tion range of 0.1–1.0 ppb and 50–950 ppb with an incubation time of 10 min and 4 min. The constructedsol–gel biosensor requires less time for analysis and no preconcentration/extraction was needed.

� 2011 Elsevier B.V. All rights reserved.

1. Introduction

Pesticides widely used throughout the world are highly toxic andtheir remanence in the environment poses serious problems due tolong-term exposure. Among the existing pesticides the organophos-phorous (OP) and carbamate insecticides represent an importantclass of toxic compounds; their toxicity can be estimated based onthe inhibition of acetyl-cholinesterase (AChE). Despite the fact thatthe organophosphorous and carbamate insecticides are not as per-sistent into the environment as the organochlorine insecticides,their intensive use has led to the contamination of ground water[1,2] and food samples [3,4].

Methyl parathion (MP), monocrotophos (MCP) are organophos-phorus insecticides (Fig. 1) and they are widely used in agriculturalor in forestry practices. The misuse of these pesticides results incontamination of fields, crops, water and air. Both the compoundsare relatively insoluble in water, poorly soluble in petroleum ether

and mineral oils, and readily soluble in most organic solvents. Thedistribution of these pesticides in air, water, soil and organisms inthe environment is influenced by several physical, chemical andbiological factors. These pesticides are readily absorbed via allroutes of exposure (oral, dermal, inhalation) and are rapidly dis-tributed to the tissues of the body. MP and MCP are cholinesteraseinhibitors and are more toxic.

Several analytical methods are developed to detect neurotoxicpesticides in various matrices. Routine methods depends uponextraction, clean up and analysis, which are normally performedby in gas chromatography (GC), high performance liquid chroma-tography (HPLC), liquid chromatography (LC) coupled to sensitiveand specific detectors such as nitrogen–phosphorous detectors(NPD) [5–7], flame ionization detectors (FID), ultraviolet detectors(UV) or diode array detector (DAD) [8–10] and mass spectrometry(MS) [11,12]. These methods are highly sensitive, capable to deter-mine a large number of compounds but they are costly, requiresmore time and complex. Numerous enzymatic electrochemicalsensors [13–16] have been developed and emerged as a powerfultool for analyzing different biological substances and organic com-pounds [17]. The more usage of electrochemical biosensors wasdue to their simple preparation by modifying the electrode surface

1572-6657/$ - see front matter � 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.jelechem.2011.11.020

⇑ Corresponding authors. Tel.: +91 877 2289303 (T. Madhusudana Reddy), +918282 256228 (B.E. Kumara Swamy).

E-mail addresses: tmsreddysvu@gmail.com (T. Madhusudana Reddy), kumaraswamy21@yahoo.com (B.E. Kumara Swamy).

Journal of Electroanalytical Chemistry 665 (2012) 76–82

Contents lists available at SciVerse ScienceDirect

Journal of Electroanalytical Chemistry

journal homepage: www.elsevier .com/locate / je lechem

with substances for selective biochemical recognition. Many trans-ducers have been constructed for biochemical sensing such as opti-cal and piezoelectric. These biosensor devices appeared as usefulalternatives to conventional methods for the detection of differentanalytes.

Among the electrochemical sensors, enzyme modified elec-trodes have gained more importance and they have been appliedeither directly or indirectly using inhibitor effects of hazardouscompounds on the enzyme activities. The biosensors approachbased on inhibition mechanism of cholinesterase enzymes (ChEs)can be advantageously used for carbamate and organophosphorouspesticide determinations [18].

In the development of biosensors the essential point was theimmobilization of the biological receptor in an active form ontothe electrode surface. The Ref. [19] has reported different immobi-lization procedures, which were used in the fabrication of screen-printed electrodes in various configurations. Among several en-zyme immobilization procedures, simple adsorption, suffers fromthe poor stability of the biosensor due to the enzyme leaking[20], covalent binding with bifunctional reagents such as glutaral-dehyde on one hand allows a stable attachment and quick re-sponse time but on the other hand gives poor reproducibility andsignificant loss of enzyme activity [21–23]. The Ref. [24] has re-ported enzyme immobilization by electropolymerization, resultingan extremely simple and rapid one-step procedure for the determi-nation of pesticides. This method can affect the denaturation of theenzyme during immobilization process. An AChE biosensor hasbeen developed for the determination of oxydemeton methyl pes-ticide by voltammetric method [25]. A simple method has beenproposed to immobilize AChE on silica sol–gel (SiSG) film with goldnanoparticles (AuNPs) for the determination of MCP by ampero-metric technique [26].

In this work, the silica sol–gel entrapped with AChE onto thecarbon paste electrode was used as a working electrode for thedetermination of MP and MCP in spiked water and fruit samples.The AChE inhibition occurs towards the fruit and water sampleswithout spiking the pesticide residues (matrix effect), and the pro-cedure to minimize the matrix effect was presented. The developedbiosensor method proved to be sensitive enough for applying inthe analysis of water and fruit samples.

2. Experimental

2.1. Materials

All chemicals were obtained from commercial sources and usedwithout further purification. Acetyl cholinesterase (E.C. 3.1.1.7type-VI-S/1.5 mg, electric eel source, 500 U/1.5 mg), acetylthiocho-line chloride were purchased from Sigma-Aldrich chemicals Co.USA. MP and MCP were obtained from Riedel-deHaen, Fluka,USA. The pesticide stock solution was prepared in acetone (GRgrade solution was obtained from Merck specialties Pvt. Ltd.). Tet-raethyl orthosilicate (TEOS), cetyltrimethyl ammonium bromide(CTAB), Triton-X-100 were obtained from Sigma-Aldrich chemicalsCo. USA. The graphite fine powder was procured from Lobo Chemieand silicon oil from Himedia. Phosphate buffer solution (PBS) wasprepared by mixing 0.1 M sodium dihydrogen phosphate monohy-drate and 0.1 M disodium hydrogen phosphate. All the aqueoussolutions were prepared by using double distilled water. The en-zyme stock solution was stored at �20 �C. All stock and workingsolutions of chemicals were stored at �4 �C.

2.2. Apparatus

The electrochemical experiments were carried out using a CH –Electrochemical Analyzer (model CH-660C, CH Instruments, USA).All the experiments were carried out in a conventional three elec-trode set up with the sol–gel immobilized enzyme electrode bodyas a working electrode, platinum (Pt) wire as an auxiliary electrodeand saturated calomel electrode as reference electrode. All theexperiments were carried out at room temperature 25 ± 2 �C.

2.3. Preparation of bare carbon paste electrode

The bare carbon paste electrode was prepared by hand mixingof 70% graphite powder and 30% silicon oil in an agate mortar toproduce a homogenous carbon paste. The paste was packed intothe cavity of homemade PVC (3 mm in diameter) and thensmoothed on a weighing paper. The electrical contact was provided

Fig. 1. The structure and molecular formulae of monocrotophos and methyl parathion.

P. Raghu et al. / Journal of Electroanalytical Chemistry 665 (2012) 76–82 77

by copper wire connected to the paste at the end of the tube[27–29].

2.4. Fabrication of biosensor

2 ml of TEOS, 1 ml of H2O, 50 ll of 0.1 M HCl, 25 ll of 10% Tri-ton-X-100 were magnetically stirring for 1 h until a homogenousTEOS silica sol–gel was obtained. The mixture was homogenizedbefore each usage and stored at �20 �C for two months.

The 5 ll of stock sol–gel solution was vortexed with 45 ll ofphosphate buffer containing 0.5 U of enzyme stock solution andfrom this 4 ll of the enzyme sol of concentration 0.04U was spreadon the electrode surface. This film was allowed to polymerize atroom temperature for about 3–5 min. This electrode was gentlywashed with phosphate buffer solution (pH 7.0) and was usedfor further experimental procedures [25].

2.5. Sample preparation

The environmental and food samples prone to be contaminatedwith pesticides were taken for study. Water samples (tap waterand mineralized water) which are used for drinking purpose wereemployed directly for the study. The fruit samples were taken froma field of Badra Reservoir Project (Karnataka, India). The fruits werewashed with distilled water and chopped. Five grams of eachchopped sample were added with 10 ml of PBS (pH 7.0) and stirredfor 1 h at room temperature. The sample solutions were filteredand samples were dissolved in acetone. These samples were spikedwith 0.4 ppb, 0.2 ppm concentration of MP and MCP, respectively.

3. Results and discussion

3.1. Voltammetric characterization of biosensor

The cholinesterase as a bio recognition element enables thedetection of toxic compounds such as organophosphorous and car-bamate pesticides. The enzymatically catalyzed hydrolysis of acet-ylthiocholine was evaluated by using the fabricated AChE-biosensor by cyclic voltammetry in 0.1 M PBS at pH 7.0. At theseexperimental conditions thiocholine was produced and its anodicoxidation gives rise to anodic peak around 0.60 V versus saturatedcalomel electrode. Fig. 2A shows the cyclic voltammograms of thesensor in the absence and presence of the substrate (1–5 mM) inPBS (pH 7.0) at the scan rate of 10 mV s�1. The effect of substrateconcentration on the response of the biosensor was shown inFig. 2B. The kinetics of the immobilized enzyme showing Michae-lis–Menten plot characteristics, was diphasic in nature, i.e. at lowamounts of the substrate, the enzyme rate was varied with concen-tration (first order) and at high concentrations of substrate, therate was independent of concentration (zero order). The apparentMichaelis–Menten constant (Kapp

m ) was 0.45 mM which was ob-tained from the linear portion of the calibration plot using Linewe-aver–Burk equation [30].

3.2. Effect of pH and enzyme concentration

The electrochemical response of the substrate at AChE-biosen-sor was generally a pH dependent and had shown in Fig. 3A. Theenzyme electrode shows maximum sensitivity at pH 7.0. The ano-dic peak potentials were shifted to less positive side with increas-ing in the pH values were depicted in Fig. 3B. The graph has a goodlinearity with a slope of 52 mV/pH. This slope value was close tothe theoretical slope of (59 mV/pH) which his in accordance withNernst equation for transfer of equal number of electrons and pro-tons in the reaction [27,28,31,32].

Fig. 3C shows the effect of enzyme concentration on measuringthe anodic peak oxidation current of thiocholine. All the experi-ments were carried out in 1 mM substrate solution prepared in0.1 M PBS. The amount of enzyme used during the process ofentrapment in sol–gel matrix was increased between 0.03 U to0.3 U, it was noticed that the response of biosensor increases cor-respondingly. Accordingly 0.04 U of enzyme was chosen as an opti-mal enzyme immobilization for the fabricated biosensor due to thefact that it helps in attaining a high percentage of inhibition of pes-ticides under low concentrations.

3.3. Detection of pesticides using AChE-biosensor

The AChE biosensor construction could potentially provideimportant information about the relative inhibitory effect of MPand MCP pesticides. An incubation time of 10 and 4 min, respec-tively were selected as a good compromise between the require-ment for a rapid assay and an achievement of high degree ofinhibition for MP and MCP respectively. The AChE biosensors weretested using standard solutions of MP and MCP pesticides. All stan-dard solutions of pesticides were freshly prepared daily to avoiddecomposition.

The pesticide determination was carried out in a three step pro-cedure. The electrode response was first measured in 0.1 M phos-phate buffer, 0.1 M KCl, pH 7.0 and 1 mM substrate. This valuecorresponded to Ii, the current before inhibition. The electrode

Fig. 2. (A) Cyclic voltammogram of enzyme electrode in 0.1 M phosphate buffer, pH7.0 and 0.1 M KCl (a) without substrate (b–f) with 1–5 mM substrate. (B)Calibration graph of substrate obtained with AChE based biosensor in 0.1 Mphosphate buffer/0.1 M KCl at pH 7.0.

78 P. Raghu et al. / Journal of Electroanalytical Chemistry 665 (2012) 76–82

was then washed with the same buffer and incubated in pesticidesolution at a known concentration. After incubation, the electroderesponse was measured, this second value corresponds to IF, thecurrent after inhibition. The percentage of enzyme inhibition andresidual enzyme activity percentage was determined accordingto the following formula [33].

% Inhibition ðI%Þ ¼ ½ðIi � IFÞ=Ii� � 100 ð1Þ

% Residual enzyme activity ðREA%Þ ¼ ½IF=Ii� � 100 ð2Þ

The inhibitory effect of different pesticides on AChE biosensorswas evaluated by determining the decrease in the current obtainedfor the oxidation of thiocholine that was produced by the enzymes.The detection of individual pesticides was carried out according to

the above procedure. Calibration plots based on the dependence ofthe% inhibition versus concentration were linear and the same wasshown in the Fig. 4A and B for MP and MCP, respectively. The limitsof detection and quantification values were 0.04 ppb, 47 ppb and0.14 ppb, 158 ppb for MP and MCP, respectively. The lowest Limitof Detection (LOD) value was achieved in the case of MP, whichwas most toxic than MCP. The variation of residual enzyme activitywith incubation time at different inhibitor concentrations wasshown in the Fig. 4C and D for MP and MCP, respectively. Fig. 4Eand F shows the variation of residual enzyme activity with concen-tration of MP and MCP, respectively. Limit of Detection (LOD) andLimit of Quantification (LOQ) studies were carried out by using thefollowing expressions [34–37].

LOD ¼ 3Sb=S ð3Þ

LOQ ¼ 10Sb=S ð4Þ

where Sb is the standard deviation of mean values for ten cyclic vol-tammograms of blank solution, S is the slope of the working curve,LOD is the limit of detection and LOQ is the limit of quantification.Table 1 shows the various parameters determined for MP and MCP.

The robustness of the developed method was evaluated bystudying the concept of repeatability (new electrode, same stan-dard solution, same day, same analyst, ‘n’ is number of assays)and reproducibility (new electrode, new standard solution, differ-ent days, different analyst, ‘n’ is number of assays) of the biosensortowards the inhibition of pesticides [34,35]. To study this experi-ments the chosen concentration of the stock solutions of MP andMCP were 0.4 ppb and 450 ppb. The results obtained for the devel-oped procedure towards the inhibition of both the pesticides wasreproducible, because there was no significant difference betweenthe RSD values of the both the pesticides. The results were shownin Table 1. The long term stability of the AChE biosensor was inves-tigated under the storage conditions (at 4 �C), it was noticed thatthe activity of immobilized AChE was stable up one month.

3.4. Determination of pesticides in water and fruit samples

Table 2 shows % of AChE inhibition for water and fruit samples.Different inhibition values were observed depending on the sam-ples analyzed. However, the water and fruit samples showed aninitial matrix effect and this was minimized by subtracting the ma-trix effect value against the value obtained for the samples spikedwith pesticide residues. Percentages of inhibition for unspikedsamples (matrix effect) may occur mainly due to the denaturingconditions (pH) or presence of ascorbic acid [38]. Table 2 showsdifferences from 15.5% to 63% which were obtained dependingupon the samples. The higher is the difference the lower is the ma-trix effect, as observed for mineralized water. High matrix effectswere observed for orange giving a difference of only 15.5%. The dif-ference between blank values and spiked values for the rest of thesamples has given values higher than 30%. Therefore the biosensorwas able to screen easily the water and fruit samples containingthe pesticides MP and MCP. All measurements were performed intriplicate. The recovery efficiencies (R%) for the different systemswere calculated using the following equation.

%R ¼ 100 ½pesticide� found=½pesticide� expectedð Þ ð5Þ

The precision and accuracy of methodologies was tested withdifferent standard solutions of each pesticide and the relative stan-dard deviations (RSD) were calculated using the followingequation.

RSD ¼ Sb=X ð6Þ

where ‘Sb’ is standard deviation of mean inhibition values obtained

and ‘X’ is the mean inhibition [39]. The results obtained in Table 2

Fig. 3. (A) Effect of pH on the enzyme electrode response. (B) Plot of Epa versus pHto 1 mM substrate. (C) The response of the biosensor varying with the concentrationof enzyme in sol–gel matrix.

P. Raghu et al. / Journal of Electroanalytical Chemistry 665 (2012) 76–82 79

conclude that, when the pesticide solution was spiked to the fruitsamples the percentage of inhibition was lower in comparison withthe sum of the inhibition caused by the spiked pesticide and matrix

effect. The detection limit of various electro analytical/other meth-ods proposed for the determination of MP and MCP was comparedwith our analytical data in Table 3. From the data shown, LOD canbe achieved using the proposed method [26,40–46].

4. Conclusions

AChE based biosensor was found to be capable in determiningorganophosphorous pesticides in different spiked samples. Thesystem showed to be successful in screening of MP and MCP pesti-cides in water and fruit samples. The use of silica sol–gel solutionas supporting material for the immobilization of enzyme, requireslow amount and the use of simple solutions which allowed the bio-sensor to operate at a relatively low cost. The advantages of thisbiosensor were simple to construct, easy to operate, samples canbe tested without any preconcentration steps and they can easilybe applied to the field monitoring.

Fig. 4. (A) Inhibition curves of immobilized AChE by methyl parathion after 10 min of incubation time. (B) Monocrotophos after 4 min incubation time. Measurementconditions: 0.1 M phosphate buffer/0.1 M KCl, pH 7.0 and 1 mM substrate. (C) and (D) Effect of incubation time on various inhibitor concentrations in 0.1 M phosphate buffer/KCl pH, 7.0.for methyl parathion and for monocrotophos respectively. (E) and (F) The variation of residual enzyme activity with different inhibitor concentrations in 0.1 Mphosphate buffer/KCl, pH 7.0 for methyl parathion and for monocrotophos, respectively.

Table 1The various parameters determined for methyl parathion and monocrotophos.

Parameters Monocrotophos Methylparathion

Response time (min) 1 1Incubation time (min) 4 10Linear range 50–950 ppb 0.1–1.0 ppbIntercept of calibration curve 32.4 19.7Slope of calibration curve 0.0737 78.83Correlation coefficient 0.9946 0.9955Standard deviation of calibration curve 3.3029 3.3258Limit of Detection (LOD) 47 ppb 0.04 ppbLimit of Quantification (LOQ) 158 ppb 0.14 ppbRepeatability (%RSD) (n = 3) 2.51 3.07Reproducibility (%RSD) (n = 3) 3.25 3.86

n = Number of assays.

80 P. Raghu et al. / Journal of Electroanalytical Chemistry 665 (2012) 76–82

Acknowledgments

The authors are very much thankful to the Department of Sci-ence and Technology (DST), Government of India, New Delhi forthe funding given through Project No. SR/FT/CS-025/2009.

References

[1] M.P. Garcia deL Lasera, M. Bernal-Gonzalez, Water Res. 35 (2001) 1933–1940.[2] T.A. Albanis, D.G. Hela, T.M. Sakellarides, I.K. Konstantino, J. Chromatogr. A 823

(1998) 59–66.[3] D.F. Rawn, V. Roscoe, T. Krakalovich, C. Hanson, Food Addit. Contam. 21 (2004)

55.[4] C. Blasco, M. Fernandez, A. Pena, C. Lino, M.I. Silveira, G. Font, Y. Pico, J. Agric.

Food Chem. 51 (2003) 8132–8138.[5] S. Lacorte, C. Molina, D. Barcelo, Anal. Chim. Acta 281 (1993) 71–84.[6] Y. Pico, A.J. Louter, J.J. Vreuls, U.A. Brinkman, Analyst 119 (1994) 2025.[7] E. Ballesteros, M.J. Parrado, J. Chromatogr. A 1029 (2004) 267–273.[8] S. Lacorte, D. Barcelo, J. Chromatogr. A 725 (1996) 85–92.[9] J. Norberg, J. Slobodnik, R.J.J. Vreuls, U.A.T. Brinkman, Anal. Methods Instrum. 2

(1995) 266.[10] V. Pichon, M.C. Hennion, J. Chromatogr. A 665 (1994) 269–281.[11] M. Albareda-Sirvent, A. Merkoci, S. Alegret, Anal. Chim. Acta 442 (2001) 35–44.[12] M.P. Marco, D. Barcelo, Meas. Sci. Technol. 7 (1996) 1547.[13] T.E. Edmonds, Chimical Sensors, fourth ed., Blackie, Glass gow, 1988.[14] P. Hauptmann, 1991 Sensors-principles and Application, Carl Hanser,

Munchen, Prentice Hall, Hamel Hempstead, 1993.[15] W. Gopel, T.A. Jones, M. Kleitz, J. Lundstrom, T. Seiyama, (Eds.), Chemical and

biochemical sensors, part I, II, W. Gopel, J. Hesse, J.N. Zemel, (Eds.), Sensors – AComprehensive Survery, vols. 2 and 3, 1991, 1992, VCH, Winheim.

[16] M. Lambrechts, W. Sansen, Biosensor: Micro Electrochemical Devices, Instituteof physics. Publ., Bristol, 1992.

[17] K.A. Hassal, The chemistry of pesticides: their metabolism, Mode of Action andUses of Crop Protection, Mac Millan, New York, 1983.

[18] D. Barcelo, M.C. Hennion, Trace Determination and Their Degradation Productsin Water, vol. 19, Elsevier, Amsterdam, 1997. pp. 544.

[19] M. Albareda-Sirvent, A. Merkoci, S. Alegreat, Sens. Actuators B 69 (2000)153–163.

[20] M.H. Gil, A.P. Piedade, S. Alegret, J. Alonso, E. Martinez-Fábregas, A. Orellana,Biosens. Bioelectron. 7 (1997) 645–652.

[21] J. Kulys, E.J. D’Costa, Biosens. Bioelectron. 6 (1991) 109–115.[22] Y.-G. Li, Y.-X. Zhou, J.-H. Jiang, L.-R. Ma, Anal. Chim. Acta 382 (1999) 277–282.[23] P. Skladal, M. Fiala, J. Krejci, Int. J. Environ. Anal. Chem. 65 (1996) 139–148.[24] T.J. Bachmann, R.D. Schmid, Anal. Chim. Acta 401 (1999) 95–103.[25] K. Anitha, S. Venkata Mohan, S. Jayarama Reddy, Biosens. Bioelectron. 20

(2004) 848–856.[26] D. Du, S. Chen, J. Cai, A. Zhang, Biosens. Bioelectron. 23 (2007) 130–134.[27] Umesh Chandra, B.E. Kumara Swamy, Ongera Gilbert, B.S. Sherigara,

Electrochem. Acta 55 (2010) 7166–7174.[28] Ongera Gilbert, B.E. Kumara Swamy, Umesh Chandra, B.S. Sherigara, J.

Electroanal. Chem. 636 (2009) 80–85.[29] S. Chithravathi, B.E. Kumara Swamy, Umesh Chandra, G.P. Mamatha, B.S.

Sherigara, J. Electroanal. Chem. 645 (2010) 10–15.[30] S. Andreescu, L. Barthelmebs, J.-L. Marty, Anal. Chim. Acta 464 (2002) 171–180.[31] B.D. Jones, J.D. Ingle Jr., Talanta 55 (2001) 699–714.[32] M.C. Shen, H.C. Gheng, V.S. Vasantha, J. Electroanal. Chem. 588 (2006)

235–243.[33] A. Hildebrandt, J. Ribas, R. Bragos, J.-L. Marty, T.C. Marius, L. Silvia, Talanta 75

(2008) 1208–1213.[34] T. Madhusudana Reddy, S.J. Reddy, Analytical Lett. 37 (2004) 2079–2098.[35] T. Madhusudana Reddy, M. Sreedhar, S.J. Reddy, J. Pharm. Biomed. Anal. 31

(2003) 811–818.[36] De Souza Djenaine, A.S. Machado Sergio, Anal. Bioanal. Chem. 382 (2005)

1720–1725.

Table 2The% inhibition of AChEs for unspiked and spiked water, fruit samples.

Sample matrix na Unspiked meaninhibition%

Pesticide Spiked Spiked meaninhibition%

(Spiked-Unspiked) value%

Expected Found Recovery(%)

Mineraliz-edwater

3 2.5 MP 0.6 ppb 65.5 63 0.62 ppb 0.59 ppb 95 ± 13 2 MCP 0.4 ppm 62 60 0.42 ppm 0.41 ppm 98 ± 1

Tap water 3 19 MP 0.6 ppb 67 48 0.69 ppb 0.64 ppb 93 ± 23 17 MCP 0.4 ppm 69 52 0.56 ppm 0.53 ppm 95 ± 1

Orange 3 47.5 MP 0.4 ppb 64.5 17 0.88 ppb 0.68 ppb 77 ± 33 51.5 MCP 0.2 ppm 67 15.5 0.69 ppm 0.50 ppm 73 ± 2

Tamota 3 22.5 MP 0.4 ppb 59.5 37 0.63 ppb 0.53 ppb 84 ± 33 25.5 MCP 0.2 ppm 75 49.5 0.44 ppm 0.37 ppm 89 ± 2

Banana 3 23.5 MP 0.4 ppb 62.5 39 0.63 ppb 0.58 ppb 92 ± 23 28 MCP 0.2 ppm 63.5 35.5 0.46 ppm 0.43 ppm 94 ± 2

a n = Number of assays.

Table 3Comparison of different electrochemical/other techniques for the determination of MP and MCP.

Biosensor Analyte (Linear Conc.range) Technique (Incubationtime)

Limit of Detection (LOD) Ref.

AChE-AuNPsa-SiSGb Monocrotophos (0.001–15 lg/ml) CV (10 min) 0.6 ng/ml [26]AChE-AuNPsa-PPyc/GCE Methyl parathion (0.019 � 10�6 to 0.45 � 10�6M) CV (12 min) 7.6 � 10�7 M [40]OPHd Methyl parathion Amperometry 20 � 10�9 M [41]Optical microbial biosensor Methyl parathion Amperometry 3 � 10�7 M [42]MPHe-Sp@AuNPsa/MWNTs/

GCEMethyl parathion (0.001–5 lg/ml) SWV 0.3 ng/ml [43]

– Methyl parathion SERSf 0.1 ppm [44]– Methyl parathion MSPDg 4 � 10�9 M [45]– Methyl parathion GC 0.04 � 10�6 M [46]AChE-SiSGb-CPE Methyl parathion (0.1–1 ppb) Monocrotophos (50–

950 ppb)CV (10 min) CV (4 min) 0.04 ppb 47 ppb Present

work

a AuNPs–Au nanoparticles.b SiSG–silica sol–gel.c PPy–polypyrrole.d OPH–organophosphorus hydrolase.e MPH–methyl parathion hydrolase.f SERS–surface enhanced Raman spectroscopy.g MSPD–matrix solid phase dispersion.

P. Raghu et al. / Journal of Electroanalytical Chemistry 665 (2012) 76–82 81

[37] Rajesh N. Hegde., B.E. Kumara Swamy, Nagaraj P. Shetti, Sharanappa T.Nandibewoor, J. Electroanal. Chem. 635 (2009) 51–57.

[38] A. Hildebrandt, R. Bragos, S. Lacorte, J.L. Marty, Sens. Actuators B 133 (2008)195–201.

[39] V.A. Pedrosa, J. Caetano, S.A.S. Machado, M. Bertotti, Sensors 8 (2008)4600–4610.

[40] Gong J. Wang, L. Zhang, Biosens. Bioelectron. 24 (2009) 2285–2288.[41] P. Mulchandani, W. Chen, A. Mulchandani, Environ. Sci. Technol. 35 (2001)

2562–2565.[42] J. Kumar, S.F. Jhask, D’. Souza, Biosens. Bioelectron. 21 (11) (2006) 2100–2105.

Epub. November 18, 2005.[43] S. Chen, J. Juang, D. Du, J.L. Li, H. Tu, D. Liu, A. Zhang, Biosens. Bioelectron. 26

(2011) 4320–4325.[44] D. Lee, S. Lee, G.H. Seong, J. bum Choo, E.K. Lee, D.-G. Gweon, S. Lee, Appl.

Spectrosc. 60 (2006) 373–377.[45] B. Albero, C.S. Brunete, J.L. Tadeo, J. Agric. Food Chem. 51 (2003) 6915–6921.[46] A.B. Muccio, P. Pelosi, I. Camoni, D.A. Barbini, R. Dommarco, T. Generali, A.

Ausil, J. Chromatogr. A 754 (1996) 497–506.

P. Raghu completed his B.Sc. degree at S.V. Arts College, Tirupati in 2007 andreceived his M.Sc. degree from the Department of Chemistry, Sri VenkateswaraUniversity in April 2009. Currently he is research scholar in the Department ofChemistry, Sri Venkateswara University, Tirupati, India. His current researchinvolves the monitoring of pesticides and its residues in environmental and foodsamples with the help of biosensors.

T. Madhusudana Reddy is a Assistant Professor in Department of Chemistry, SriVenkateswara University, Tirupati. Dr. Reddy has completed his M.Sc. degree withphysical chemistry as specialization in 1999 and Ph.D. in the year 2005 from SriVenkateswara University. Dr. Reddy teaches Electrochemistry to the P.G. students;his research mainly focuses in the field of development of chemical sensors andbiosensors and development of amperometric methods for the determination ofpesticide residues in environmental and food samples. During 2005–2007 heworked as Postdoctoral Researcher at ‘‘Chimie, Electrochimie Moléculaires et Chi-mie Analytique », Université de Bretagne Occidentale, Brest, France. He visited

France, Spain and Germany for attending conferences to present his researchresults. Currently he is running two research project funded by Department ofScience and Technology (DST) and University Grants Commission (UGC), Govern-ment of India, New Delhi, India.

B.E. Kumara Swamy received his Master of Science in Industrial Chemistry in 1997from Kuvempu University, Shimoga, Karnataka, INDIA, recipient of Prof. M.R.Gajendraghad Gold Medal and Young Scientist Award from Indian Council ofChemists in 2000 in Physical Chemistry Section and later received his Ph.D. from thesame department in August 2002. He joined as National Science Foundation (NSF)Post Doctoral Research Associate in Department of Chemistry in Southern Meth-odist University, Dallas, USA (2003–2006) and Research Associate in University ofVirginia, Virginia, USA (February 2006–December 2007). The area of interestinvolved the modification of carbon paste electrode for the detection of neuro-transmitters and carbon fiber microelectrodes. He has published more than 82papers in referred journals and at present working as Assistant Professor inIndustrial Chemistry department, Kuvempu University, Shimoga December 2006.He has guided 02 Ph.D’s, 03 M.Phil’s and presently 7 students are working for thePh.D. programme. Currently received four projects from different funding agencies.

B.N. Chandrashekar recieved his B.Sc. degree in the combination of Chemistry,Physics and Mathematics in 2005 from IDSG college, Chikkamagalure, Karnataka,India and M.Sc. in Industrial Chemistry in 2008 from Kuvempu University, Shimoga,Karnataka, India. He is currently working as a research scholar leading the award ofPh.D. degree in Kuvempu University, Shimoga, Karnataka, India. His research field ofinterest includes development of chemically modified electrode and biosensorapplications in modern analytical chemistry.

K. Reddaiah received his B.Sc. degree in 1999 and M.Sc. degree in 2009 withphysical chemistry as specialization from Sri Venkateswara University, Tirupati. Heis currently a doctoral researcher and pursuing his research in the field of chemicalsensors and chemically modified electrodes for the determination of drug andpesticide samples at department of chemistry, Sri Venkateswara University.

82 P. Raghu et al. / Journal of Electroanalytical Chemistry 665 (2012) 76–82

Anal. Bioanal. Electrochem., Vol. 5, No. 2, 2013, 139 - 153

Full Paper Development of Sol–Gel Immobilized Electrochemical Biosensor for the Monitoring of Organophosphorous Pesticides: A Voltammetric Method Pamula Raghu1, Matti Mohan Reddy2, Tukiakula Madhusudana Reddy1,*, Bahaddurghatta E. Kumara Swamy 3,* and Kasetty Reddaiah1

1Electrochemical Research Laboratory, Department of Chemistry, S.V.U. College of Sciences, Sri Venkateswara University, Tirupati-517502, Andhra Pradesh, India 2Department of Psychiatry, Sri Devaraj Ur’s Acedamy of Higher Education and Research (SDUAHER), Tamaka, Kolar, Karnataka, India 3Department of P.G. Studies and Research in Industrial Chemistry, Kuvempu University, Shankaraghatta -577 451, Shimoga, Karnataka, India *Corresponding Author, Tel.:+91-877-2289303; Fax: +91-877-2248499/2249111

E-Mails: tmsreddysvu@gmail.com, kumaraswamy21@yahoo.com

Received: 27 January 2013 / Accepted: 13 April 2013 / Published online: 30 April 2013

Abstract- Acetylcholinesterase (AChE) enzyme was immobilized through the silica sol–gel process onto the surface of carbon paste electrode (CPE). This fabricated monoenzyme biosensor on CPE was used as a working electrode. The enzyme biosensor on reaction with acetylthiocholine chloride (ASChCl or substrate), was found to be enzymatically hydrolyzed to thiocholine and acetic acid, which intern gave a disulfide compound and produced a larger anodic current at 0.63 V. The AChE biosensor was used for determining the two organophosphorous pesticides i.e. quinalphos and malathion in 0.1 M phosphate buffer/0.1 M KCl. The effect of scan rate, pH, enzyme loading and substrate concentration on the biosensor response was studied. Calibration graphs were performed for a concentration range of 20–300 ppb and 0.07–1.3 ppm for quinalphos and malathion respectively by employing the fabricated biosensor electrode. The limit of detection and limit of quantification values was found to be 8 ppb, 0.058 ppm and 26 ppb, 0.194 ppm for quinalphos and malathion respectively. Keywords- Biosensor, Acetylcholinesterase, Acetylthiocholine Chloride, Sol–Gel Immobilization, Quinalphos and Malathion

Analytical & Bioanalytical Electrochemistry

© 2013 by CEE

www.abechem.com

Anal.Bioanal. Electrochem., Vol. 5, No. 2, 2013, 139 - 153 140

1. INTRODUCTION

Organophosphorous pesticides are widely used in agriculture because of their insecticidal

activity and their relatively low persistence in the environment. Their great success in

agricultural applications has led to an increase in the production and spread of pesticide

contamination. These pesticides are important pollutants and hazardous to human health and

life. In areas where intensive monoculture is practiced, pesticides are used as standard

method for pest control. Organophosphorous pesticides are highly effective broad spectrum

insecticides. They are increasingly used instead of organochloride pesticides due to their

lower environmental persistence. However, they present a high toxicity that may represent in

a serious rise of professional exposure and for the equilibrium of acquatic system.

Quinalphos and malathion are organophosphorus insecticides (Table 1) and they are

widely used in agricultural due to its acaricidal and insecticidal properties, especially

malathion is used in residential landscaping and in public health pest control programs such

as mosquito eradication. The misuse of these pesticides results in contamination of fields,

crops, water and air. Both the compounds are relatively insoluble in water, poorly soluble in

petroleum ether and mineral oils, and readily soluble in most organic solvents. These

pesticides represent a source of toxicity towards human beings and vertebrate animals and its

mode of action occurs through the contact and intake through food chain. Both the pesticides

are more toxic and acts as cholinesterase inhibitors.

Many methods are available for pesticide detection. Chromatographic methods such as

high performance liquid chromatography (HPLC) and gas chromatography (GC) are used as

reference methods, but they have strong drawbacks, such as complex and time consuming

treatments of the samples i.e. extraction of pesticides, extract cleaning, solvent substitution

etc. [1,2]. Moreover the analysis usually has to be performed in a specialized laboratory by

skilled personnel and is not suitable for in situ application. These issues turnout to be a major

problem when rapid and sensitive measurement is needed in order to take the necessary

corrective actions in a timely fashion. To respond to the above issues, the enzymatic methods have been adopted as an

alternative to classical methods (GC and HPLC) for faster and simpler detection of some

environmental pollutants [3].The cholinesterase based biosensors are one of the best

alternatives in the context of this strategy. These biosensors are simple to fabricate and low

cost of the equipment also make possible in situ measurement of pesticides by various

techniques such as amperometric [4-7], potentiometric [8-10] and conductometric biosensors

[11] have been developed using this approach. For amperometric detection of cholinesterase

activity the substrate acetylthiocholine chloride has been extensively used [12].

Anal.Bioanal. Electrochem., Vol. 5, No. 2, 2013, 139 - 153 141

Table 1. The structural and molecular formulae of quinalphos and malathion

Pesticide Molecular formula Structure

Quinalphos

C12H15N2O3PS

Malathion

C10H19O6PS2

Sol–gel immobilization technique can be preferred instead of usual immobilization

protocols such as covalent binding, adsorption, encapsulation of sensing agents with in a

polymeric matrix etc. Since some of these procedures are tedious, result in poor stability and

perturbed function, requiring expensive reagents or environmentally unattractive solvents. So

many sol–gel derived enzyme biosensors have been developed at the research level to

monitor glucose, lactate, phenols, urea etc. [13-16].

In this paper, we report biosensor derived from TEOS sol–gel system doped with

acetylcholinesterase towards quinalphos and malathion detection. Experimental parameters

such as the scan rate, pH and enzyme loading have been investigated to evaluate the

conditions for the best performance of the biosensor.

2. EXPERIMENTAL

2.1. Materials

Acetylcholinesterase (E.C. 3.1.1.7 type–VI–S/1.5 mg, electric eel source, 500U/1.5 mg)

and acetylthicholine chloride were purchased from Sigma–Aldrich chemicals co. USA.

Quinalphos and malathion were obtained from Accustandard solutions company, USA. The

pesticide stock solution was prepared dissolving in acetone (GR grade) solution.

Tetraethylorthosilicate (TEOS), cetyltrimethylammonium bromide (CTAB), Triton–X–100

were obtained from Sigma–Aldrich chemicals co. USA. The graphite fine powder was

procured from Lobo chemie and silicon oil from Himedia Company. The acetone (GR grade)

was obtained from Merk Specialities Pvt. Ltd. Phosphate buffer solution was prepared by

mixing 0.1 M sodium dihydrogen phosphate monohydrate and 0.1 M disodium hydrogen

phosphate. All the aqueous solutions were prepared with double distilled water. All chemicals

were obtained from commercial sources and used without further purification. The enzyme

Anal.Bioanal. Electrochem., Vol. 5, No. 2, 2013, 139 - 153 142

stock solution was stored at –20oC. All stock and working solutions of chemicals were stored

at –4 oC.

2.2. Apparatus

The electrochemical experiments were carried out using an electrochemical workstation;

model CHI–660C (CH instruments). All the experiments were carried out in a conventional

three electrode electrochemical cell. The sol–gel immobilized enzyme electrode body was

used a working electrode and Pt wire as an auxiliary electrode. To determine the potentials at

the surface of working electrode, saturated calomel electrode was used as a reference

electrode. All the experiments were carried out at room temperature (25±2 °C).

2.3. Preparation of bare carbon paste electrode

The bare carbon paste electrode was prepared by hand mixing of 70% graphite fine

powder and 30% silicon oil in an agate mortar to produce a homogenous carbon paste. The

paste was packed into the cavity of homemade PVC (3 mm in diameter) and then smoothed

on a weighing paper. The electrical contact was provided by copper wire connected to the

paste at the end of the tube [17-19].

2.4. Biosensor construction

2 ml of TEOS, 1 ml of H2O, 50 µl of 0.1 M HCl and 25 µl of 10% Triton–X–100 were

magnetically stirred for 1 hr until obtaining a homogenous TEOS silica sol. The mixture was

homogenized before each usage and stored at -20 °C. The mixture was stable for about three

months.

The 5 µl of stock solution of sol–gel was vortexed with 45 µl of phosphate buffer

containing 0.5 U of enzyme stock solution. The 5 µl of the enzyme sol was spread on the

electrode surface. This film was allowed to polymerize at room temperature for 3–5 min. This

electrode was gently washed with phosphate buffer (pH 7.0) and is used for further

experimental procedures [20]. The 0.05 U of enzyme was immobilized on the electrode

surface.

3. RESULTS AND DISCUSSION

3.1. Cyclic voltammetric characterization of biosensor

The fabricated AChE based biosensor entrapped through sol–gel immobilization method

onto the carbon paste electrode (CPE) was found to hydrolyze the substrate (acetylthiocholine

chloride), and the mechanism was shown in scheme 1. The active site of acetylcholinesterase

consists of two sub sites, namely an anionic site (a) and an esteratic site (b) which contains

the crucial serine residue. The anionic site has electrically negative potential which attracts

the quaternary ammonium head of acetylthiocholine and helps in orienting the charged part

Anal.Bioanal. Electrochem., Vol. 5, No. 2, 2013, 139 - 153 143

of the substrate entering the active center. The esteratic site is involved in the actual catalytic

process. The activated serine residue undergoes a nucleophilic attack onto the carbonyl group

present in the substrate which is present in transition tetrahedral state. The negative charge

formed on the carboxylic oxygen of acetylthiocholine was rearranged to give the products as

thiocholine and acetylated enzyme. The two molecules of thiocholine undergo oxidation

process to give a dimmer with a larger anodic peak current (Ipa) at potential 0.63 V vs.

saturated calomel electrode. The deacetylation of enzyme takes place in the presence of one

water molecule to give acetic acid and native enzyme (serine residue). Fig. 1 shows the cyclic

voltammograms of the sensor in the presence and absence of 1mM substrate in phosphate

buffer (pH 7.0) at a scan rate of 10 mV s-1. It has been reported that buffer containing

substrate electrochemically detected by using AChE [21,22]. The biosensor response varies

with addition of concentration of the substrate, first the biosensor response increases with

substrate concentration and reaches a plateau level, and it was shown in the Fig. 2.

Scheme. 1. The mechanism of the enzyme catalyzed reaction

The apparent Michaelis–Menton constant (Kmapp) was 550 µM, which was calculated

from the linear part of the calibration plot using Lineweaver–Burk equation. This data

illustrates that the immobilization was successful and AChE maintains its biological activity

within sol–gel matrix.

Anal.Bioanal. Electrochem., Vol. 5, No. 2, 2013, 139 - 153 144

Fig. 1. Cyclic voltammogram of enzyme electrode in 0.1M phosphate buffer, pH 7.0 and

0.1 M KCl (a) without substrate (dashed line) (b) with 1 mM substrate (solid line)

Fig. 2. Calibration graph for acetylthiocholine chloride obtained with AChE immobilized

sensor in 0.1 M phosphate buffer / 0.1 M KCl at pH 7.0

3.2. Effect of scan rate and pH

The effect of scan rate for 1 mM acetylthiocholine chloride on the anodic peak current

(Ipa) was studied in 0.1 M phosphate buffer solution/0.1 M KCl solution. The ‘Ipa’ increases

linearly with increasing scan rate ranging from 5 to 40 mV s-1. The graph between Ipa vs.

Anal.Bioanal. Electrochem., Vol. 5, No. 2, 2013, 139 - 153 145

square root of scan rate (υ1/2) obtained good linearity with a correlation coefficient of 0.9943

(r2), which was shown in Fig. 3. This indicates the electrode reaction was diffusion controlled

process.

Fig. 3. The enzyme electrode response by varying squareroot of scan rate for 1 mM ASChCl

in 0.1 M phosphate buffer (pH 7.0) and 0.1 M KCl

The effect of pH on the biosensor response towards the 1 mM acetylthiocholine chloride

over the pH range 5.5 to 8.5 in 0.1 M KCl solution was shown in Fig. 4A. As the pH of the

solution increases the response of the biosensor increases until attaining a physiological pH

7.0 and there onwards the response of the biosensor decreases. The enzyme electrode shows

maximum sensitivity at pH 7.0. The anodic peak potentials (Epa) shifts to less positive side

potentials with increasing pH of the buffer solution. The graph of Epa vs. pH of the solution

was shown in Fig. 4B. It shows good linearity with a slope of 53 mV/pH. This behavior

nearly obeys the Nernst equation for equal number of electrons and protons transfer reaction

[23,24]. All experiments including inhibition studies were carried out at pH 7.0.

Anal.Bioanal. Electrochem., Vol. 5, No. 2, 2013, 139 - 153 146

Fig. 4. (A) Effect of the pH on the enzyme electrode response (B) Plot of Epa vs. pH to 1 mM

ASChCl

3.3. Effect of enzyme loading

Fig. 5 shows the study of influence of the amount of enzyme added to the electrode on the

current response measured as a consequence of the anodic oxidation of generated thiocholine.

Experiments were carried out in 1 mM acetylthiocholine chloride solution prepared in 0.1 M

phosphate buffer. The amount of enzyme immobilized onto the CPE was varied between 0.03

to 0.3 U. The biosensor response increases with increasing in the concentration of the

enzyme. Accordingly, 0.05 U of acetylcholinesterase was chosen as the optimal enzyme

A

B

Anal.Bioanal. Electrochem., Vol. 5, No. 2, 2013, 139 - 153 147

immobilization solution for the fabricated biosensor due to the fact that it helps in attaining a

high percentage of inhibition of pesticides under low concentrations [11].

Fig. 5. The effect of enzyme loading into sol–gel matrix on the response of biosensor system

3.4. Pesticide detection studies

The monoenzyme biosensor was used for the measurement of quinalphos and malathion.

The sensor was used to carry out inhibition studies by incubating with pesticide solution up to

4 min to obtain lower detection limits. Sol–gel immobilization method could provide wider

concentration range of pesticide detection sensitive enough up to ppb level. The biosensor

entrapped through sol–gel immobilization technique onto the CPE was dipped into the

electrolytic cell containing 5 ml of 0.1 M phosphate buffer solution and 1 mM

acetylthiocholine chloride (substrate), the signal originated from the enzyme–substrate

reaction was recorded (Ii). The enzyme inhibition study was carried out by adding the

pesticide samples of various concentrations into the electrolytic cell. The electrochemical

measurement was recorded for every addition of pesticide sample and the corresponding

pesticide inhibition was recorded (IF). The enzyme inhibition was found to be proportional to

the concentration of pesticide solution. The enzyme inhibition percentage and residual

enzyme activity percentage was calculated using the following equations [25].

Anal.Bioanal. Electrochem., Vol. 5, No. 2, 2013, 139 - 153 148

Inhibition % ( I% ) = [ (Ii – IF) / Ii ] X 100 (1)

Residual enzyme activity % (REA %) = [ IF / Ii ] X 100 (2)

The organophosphorous compounds inhibit the enzymatic hydrolysis reaction which in

turn decreases the concentration of thiocholine and leads to the decreases in anodic peak

current with increasing inhibition. The detection of individual pesticides was carried out

according to the above procedure. Calibration plots based on the dependence of the %

inhibition vs. concentration was linear and the same was shown in Fig. 6A&B for quinalphos

and malathion respectively. The behavior of enzyme activity within the concentration range

of 20–300 ppb and 0.07–1.3 ppm at different incubation times was shown in Fig.7A&B for

quinalphos and malathion respectively. It reveals that the level of inhibition of enzyme

increases with increase in incubation time and as well as increase in concentration of

pesticides. A complete inhibition was observed at shortest incubation time of 4 min for

quinalphos and malathion of concentrations 300 ppb&1.3 ppm respectively. Fig. 8 shows the

differential pulse voltammograms of substrate alone and pesticide solution, where complete

inhibition occurred. As the concentration of pesticides increases the residual enzyme activity

of the enzyme decreases with respect to time and same was shown in the Fig. 9A&B for

quinalphos and malathion respectively. The determination of LOD and LOQ was carried out

by using the following expression [17,26-28].

LOD = 3Sb/S (3)

LOQ = 10Sb/S (4)

Where ‘Sb’ is standard deviation of the mean values for ten voltammograms of the blank

solution, ‘S’ is the slope of the working curve, LOD is the limit of detection and LOQ is the

limit of quantification. The limit of detection (LOD) and limit of quantification (LOQ) values

was found to be 8 ppb, 0.058 ppm and 26 ppb, 0.194 ppm for quinalphos and malathion

respectively. The lowest LOD value was found for quinalphos, which is more toxic than

malathion. Thus these results clearly indicate that the proposed electrochemical method of

analysis is reliable for the determination of individual pesticides. Table. 2 shows the various

parameters determined for quinalphos and malathion.

Anal.Bioanal. Electrochem., Vol. 5, No. 2, 2013, 139 - 153 149

Fig. 6. Inhibition plots of (A) Quinalphos after 4 min incubation time (B) Malathion after 4

min incubation time in 0.1 M phosphate buffer (pH 7.0)/0.1 M KCl

Fig. 7. Effect of incubation time on residual enzyme activity for various inhibitor

concentrations in 0.1M phosphate buffer / KCl pH 7.0 (A) For Quinalphos (a) 20 ppb (b) 50

ppb (c) 200 ppb (d) 250 ppb (e) 300 ppb (B) For Malathion (a) 0.07 ppm (b) 0.1 ppm (c) 0.15

ppm (d) 0.40 ppm (e) 1.0 ppm (f) 1.3 ppm

ABB

Anal.Bioanal. Electrochem., Vol. 5, No. 2, 2013, 139 - 153 150

Fig. 8. Differential pulse voltammograms of (a) substrate alone (b) with 300 ppb of

quinalphos/1.3 ppm of malathion pesticide solution

Fig. 9. The variation of residual enzyme activity with different inhibitor concentrations with

respect to time in 0.1 M phosphate buffer / 0.1 M KCl, pH 7.0. (A) For quinalphos, (B) For

malathion

The robustness of the developed method was evaluated by studying the concept of

repeatability (new electrode, same standard solution, same day, same analyst, ‘n’ is number

of assays) and reproducibility (new electrode, new standard solution, different days, different

analyst, ‘n’ is number of assays) of the biosensor towards the inhibition of pesticides [26,27].

To study this experiment the chosen concentration of the stock solutions of quinalphos and

malathion were 100 ppb and 0.5 ppm. The results obtained for the developed procedure

towards the inhibition of both the pesticides was reproducible, because there was no

A B

Anal.Bioanal. Electrochem., Vol. 5, No. 2, 2013, 139 - 153 151

significant difference between the RSD values of the both the pesticides. The results were

shown in Table 2. The long term stability of the AChE biosensor was investigated under the

storage conditions (at 4°C), it was noticed that the activity of immobilized AChE was stable

up one month.

Table 2. The various parameters determined for quinalphos and malathion

(n = number of assays)

4. CONCLUSION

The present investigation demonstrates the electrochemical detection of pesticides by using

acetylthiocholine chloride as a substrate. The thiocholine can be electrochemically detected at

sol – gel immobilized carbon paste electrode, through direct oxidation of these analytes at

slightly more anodic potential 0.63 V vs. saturated calomel electrode. The immobilization of

enzyme is much simpler and generates good results. The fabricated electrochemical biosensor

is of fast response, adequate reproducibility, large pesticide working ranges and sensitive to

the determination of organophosphorous pesticides.

Acknowledgements

The authors are gratefully acknowledging the Department of Science and Technology

(DST), Government of India, New Delhi, for the financial support received through the fast

track research project no. SR/FT/CS -025/2009.

Parameters Quinalphos Malathion

Response time (min) 1 1

Incubation time (min) 4 4

Linear range 20 – 300 ppb 0.07 – 1.3 ppm

Intercept of calibration curve 4.1902 23.5756

Slope of calibration curve 0.3020 62.1014

Correlation coefficient 0.9949 0.9905

Standard deviation of

calibration curve

3.8482 5.0397

Limit of detection (LOD) 8 ppb 0.058 ppm

Limit of quantification (LOQ) 26 ppb 0.194 ppm

Repeatability (%RSD) (n=3) 2.54 3.72

Reproducibility (%RSD) (n=3) 3.11 4.29

Anal.Bioanal. Electrochem., Vol. 5, No. 2, 2013, 139 - 153 152

REFERENCES

[1] I. Liska, and J. Slobodnik, J. Chromatogr. A 733 (1996) 235.

[2] S. Lacorate, and D. Barcelo, Anal. Chem. 68 (1996) 2464.

[3] A. Amine, H. Mohammadi, I. Bourais, and G. Palleschi, Biosens. Bioelectron. 21

(2006) 1405.

[4] A. L. Hart, W. A. Collier, and D. Janssen, Biosens. Bioelectron. 12 (1997) 645.

[5] M. Bernabei, S. Chiavarini, C. Cremisini, and G. Palleschi, Biosens. Bioelectron. 8

(1993) 265.

[6] M. Trojanovic, and M. L. Hitchman, Trends. Anal. Chem. 15 (1996) 38.

[7] E. V. Gogol, G. A. Evtyugin, E. V. Suprun, G. K. Budnikov, and V. G. Vinter, J. Anal.

Chem. 56 (2001) 963.

[8] K. Reybier, S. Zairi, N. Jaffrezic-Renault, and B. Fahys, Talanta 56 (2002) 1015.

[9] A. N. Ivanov, G. A. Evtugyn, R. E. Gyuresanyi, K. Toth, and H. C. Budnikov, Anal.

Chim. Acta 404 (2000) 55.

[10] T. Imato, and N. Ishibashi, Biosens. Bioelectron. 10 (1995) 435.

[11] S. Suwansaard, P. Kanatharana, P. Asawatreratanakul, C. Limsakul, B.

Wongkittisuksaa, and D. P. Thavarungkul, Biosens. Bioelectron. 21 (2005) 445.

[12] M. Trojanowicz, Electroanalysis 14 (2002) 1311.

[13] S. Sampth, and O. Lev, Anal. Chem. 68 (1996) 2015.

[14] T. M. Park, E. I. Iwuoha, M. R. Smyth, R. Freoney, and A. J. Mcshane, Talanta 44

(1997) 973.

[15] J. Li, L. S. Chia, N. K. Goh, and S. N. Tan, Anal. Chim. Acta 362 (1998) 203.

[16] P. C. Pandey and G. Singh, Talanta 55 (2001) 773.

[17] C. Umesh, B. E. Kumara Swamy, G. Ongera, and B. S. Sherigara, Electrochim. Acta

55 (2010) 7166.

[18] G. Ongera, B. E. Kumara Swamy, C. Umesh, and B. S. Sherigara, J. Electroanal.

Chem. 636 (2009) 80.

[19] S. Chithravathi, B. E. Kumara Swamy, C. Umesh , G. P. Mamatha, and B. S. Sherigara

J. Electroanal. Chem. 645 (2010) 10.

[20] K. Anitha, S. Venkata Mohan, and S. Jayarama Reddy, Biosens. Bioelectron. 20

(2004) 847.

[21] L. H. Goodson, and W. B. Jacobs, Enzymol 44 (1976) 647.

[22] R. Gruss, F. Scheller, M. J. Shao, and C. C. Liu, Anal. Lett. 22 (1989) 1159.

[23] B. D. Jones, and J. D Ingle. Jr, Talanta 55 (2001) 699.

[24] M. C. Shen, H. C. Gheng, and V. S. Vasantha, J. Electroanal. Chem. 588 (2006) 235.

[25] A. Hildebrandt, J. Ribas, R. Bragos, J. L. Marty, M. Tre- sanchez, and S. Lacorte

Talanta 75 (2008) 1208.

Anal.Bioanal. Electrochem., Vol. 5, No. 2, 2013, 139 - 153 153

[26] T. Madhusudana Reddy, and S. J. Reddy, Anal. lett. 37 (2004) 2079.

[27] T. M. Reddy, M. Sreedhar, and S. J. Reddy, J. Pharm. Biomed. Anal. 31 (2003) 811.

[28] R. N. Hegde, B. E. Kumara Swamy, N. P. Shetti, and S. T. Nandibewoor, J.

Electroanal. Chem. 635 (2009) 51.

Copyright © 2013 by CEE (Center of Excellence in Electrochemistry)

ANALYTICAL & BIOANALYTICAL ELECTROCHEMISTRY (http://www.abechem.com)

Reproduction is permitted for noncommercial purposes.

Anal. Bioanal. Electrochem., Vol. 4, No. 1, 2012, 1 - 16

Full Paper

Detection of Organophosphorous Pesticides Using a Monoenzyme Biosensor: A Voltammetric Study

Pamula Raghu1, Matti R. Mohan Reddy 2, Tukiakula M. Reddy 1,*, Bahaddurghatta E. Kumara Swamy 3,* and Kasetty Reddaiah 1

1Electrochemical Research Laboratory, Department of Chemistry, S.V.U. College of Sciences, Sri Venkateswara University, Tirupati-517502, Andhra Pradesh, India 2Department of Psychiatry, Sri Devaraj Urs Acedamy of Higher Education and Research (SDUAHER), Tamaka, Kolar, Karnataka, India 3Department of P.G. Studies and Research in Industrial Chemistry, Kuvempu University, Shankaraghatta -577 451, Shimoga, Karnataka, India

*Corresponding Author, Tel: +91-877-2289303, +91-8282-256228; Fax: +91 8282 256225

E-Mails: tmsreddysvu@gmail.com , kumaraswamy21@yahoo.com

Received: 31 December 2011 / Accepted: 25 February 2012 / Published online: 28 February 2012 Abstract- Acetylcholinesterase (AChE) was an important cholinesterase enzyme present in the living organisms, which is responsible for transmission of impulses through synaptic clefts by oxidation of acetylcholine to choline. Acetylcholinesterase enzyme was immobilized through silica sol–gel process on the surface of carbon paste electrode which was used to fabricate monoenzyme biosensor. It is a rapid, simple and sensitive biosensor used for determination of two organophosphorous pesticides monocrotophos and phosphamidon in 0.1 M phosphate buffer and in 0.1 M KCl. Acetylthiocholine chloride (ASChCl) was used as substrate by enzymatic hydrolysis it gives thiocholine which undergone electrochemical oxidation and produces anodic current around at 0.60 V vs. saturated calomel electrode. The effect of scan rate, pH, enzyme loading and substrate concentration on the biosensor response was studied. The biosensor provided a high sensitivity, large linear concentration range from 50–900 ppb, 0.1–1.25 ppm for monocrotophos and phosphamidon. The detection limits were found to be 45 ppb, 0.06 ppm for monocrotophos and phosphamidon respectively. The results showed the optimum conditions for pH, substrate concentration, and incubation time were at room temperature, pH 7.0, 1 mM, 4 and 3 min for monocrotophos and phosphamidon respectively. Keywords- Acetylthiocholine Chloride, Immobilization, Acetylcholinesterase, Monocrotophos , Phosphamidon

Analytical & Bioanalytical Electrochemistry

© 2012 by CEE

www.abechem.com

Anal. Bioanal. Electrochem., Vol. 4, No. 1, 2012, 1 – 16 2

1. INTRODUCTION

Organophosphorous pesticides are widely used in agriculture and their properties provide

numerous benefits in terms of production and quality increase [1]. Their presence in water

and food poses a potential hazard to human health [2]. Hence fast and economically viable

methods are required for their detection in the environment and in agro food products [3].

Many methods have been developed in the last few years for the determination of pesticides.

The analysis of pesticides was usually carried out by gas and liquid chromatography with a

selective element detector [4, 5]. Nevertheless, these procedures are expensive and frequently

require laborious, complex, time consuming, sample treatment such as extraction of

pesticides, extract cleaning, solvent substitution and clean up steps. Furthermore these

approaches do not improve our understanding of the natural processes governing chemical

species behaviour, their transport, bioavailability, and their long term impact on aquatic

systems. The stability of samples during long–term storage was questionable, as there are

subject to various biological, chemical and physical effects [6]. Finally, the analysis was

usually performed in a specialized laboratory by skilled personnel and was not suitable for in

situ application. These issues turn out to be a major problem when rapid and sensitive

measurements are needed in order to take the necessary corrective actions in a timely

approach. Accordingly a rapid, unfailing and simple sensor for detecting pesticides continues

to be an issue of interest in electro analytical research [7, 8].

Biosensors for detection and quantification of pollutants have attracted extraordinary

interest in recent years, because of its role in the development of highly sensitive, selective,

chemical analysis, low cost and short analysis time associated with these devices. Biosensors

based on the inhibition of acetylcholinesterase have been used for the detection of pesticides

in different samples [9–11]. Different types of immobilization methods are available, among

them, sol–gel immobilization can be preferred through the usage of silicate materials for the

entrapment of a biological moiety which was introduced in the mid of 1950s [12] but

importance was not realized at that time. The entrapment of proteins into alkoxysilane–

derived silicate glasses via the sol–gel method has been reported [13]. This pioneering work

was greatly recognized by this group when the technique was applied independently to the

doping of transparent silica glasses with alkaline phosphatase, chitinase, aspartase and -

glucosidase and copper–zinc superoxide dismutase, cytochrome C and myoglobin entrapped

in tetramethyl orthosilicate [14]. Tetraethyl orthosilicate derived sol–gel monolith doped with

glucose oxidase was used as a recognition element in a flow injection analytical system [15].

The chemistry of sol–gel processing generally involves the hydrolysis of alkoxysilane or

alkyl silicate or alkoxy mettalate or a mixture of these, in the presence of acid or base

catalysis to form hydroxy derivatives. A cascade of condensation reactions gives rises to

soluble, colloidal clear sols and ultimately phase separated polymers, which produce the final

Anal. Bioanal. Electrochem., Vol. 4, No. 1, 2012, 1 – 16 3

matrices in various configurations such as monoliths, shelts, granulates, micro particles, thick

and thin films.

Accordingly, in the present work studies on the immobilization of acetylcholinesterase

enzyme onto carbon paste electrode was carried out. Experimental parameters such as the

scan rate, pH and enzyme loading have been investigated to evaluate the conditions for the

best performance of the biosensor towards the determination of pesticides. The structural and

molecular formulae of the monocrotophos and phosphamidon were shown in Table 1.

CH3

CS

N+

CH3

CH3 CH3O

+ HO

H

CH3

N+SH

CH3

CH3

+

CH3

COH

O

CH3

N+

SH

CH3

CH3

CH3

N+

S

CH3

CH3

S

N+

CH3

CH3CH3

+ 2H+ + 2e-

AChE

anodic oxidation

at 0.6V

Thiocholine

Acetylthiocholine

Dithio-bis choline

2

Scheme 1. The mechanism of the enzyme catalyzed reaction

Anal. Bioanal. Electrochem., Vol. 4, No. 1, 2012, 1 – 16 4

Table 1. The structural and molecular formulae of monocrotophos and phosphamidon

pesticides

Pesticide Molecular formula Structure

Monocrotophos C7H14NO5P

Phosphamidon C10H19ClNO5P

2 . EXPERIMENTAL

2.1 . Materials

All chemicals were obtained from commercial sources and used without further

purification. Acetylcholinesterase (E.C.3.1.1.7,type–VI–s/1.5 mg, electric eel source,

500U/1.5mg), acetylthiocholine chloride were purchased from Sigma–Aldrich chemicals,

USA. Tetraethyl orthosilicate (TEOS), cetyl trimethyl ammonium bromide (CTAB), Triton–

X-100 were obtained from Sigma–Aldrich chemicals co. USA. Monocrotophos and

phosphamidon were obtained from Accustandard solutions company, USA. The pesticide

stock solution was prepared by dissolving in acetone (GR grade) solution. The graphite fine

powder was procured from Lobo chemie and silicon oil (Himedia). The acetone (GR grade)

was obtained from Merk Specialities Pvt. Ltd. Phosphate buffer solution was prepared by

mixing appropriate quantity of 0.1 M aqueous sodium dihydrogen phosphate monohydrate

and 0.1 M aqueous disodium hydrogen phosphate. All the chemicals were of analytical grade

and aqueous solutions were prepared with double distilled water. The enzyme stock solutions

were stored at –20 oC. All stock and working solutions of chemicals were stored at – 4 oC.

2.2 . Apparatus

The electrochemical experiments were carried out using a model CH–660C

(CH Instruments, USA). All the experiments were carried out in a conventional three

electrode electrochemical cell. The electrode system contained a working electrode which

Anal. Bioanal. Electrochem., Vol. 4, No. 1, 2012, 1 – 16 5

was an enzyme modified carbon paste electrode, a platinum wire as a counter electrode and

saturated calomel electrode as reference electrode. All the experiments were carried out at

room temperature 25±2 oC.

2.3 . Preparation of bare carbon paste electrode

The bare carbon paste electrode was prepared by hand mixing of 70% graphite powder

with 30% silicon oil in an agate mortar to produce a homogenous carbon paste. The paste was

packed into the cavity of homemade PVC (3 mm in diameter) and then smoothed on a

weighing paper. The electrical contact was provided by a copper wire connected to the paste

in the end of the tube [16-18].

2.4 . Preparation of silica sol–gel solution and enzyme modified carbon paste electrode

A homogenous TEOS silica sol was prepared by mixing 2 ml of TEOS, 1 ml of H2O,

50 l of 0.1 M HCl, 25 l of 10% triton–X-100. In this solvent less hydro sol procedure,

TEOS was added to distilled water and stirred magnetically to form a gray two phase

dispersion at room temperature. When the solution gets acidified, the dispersion transforms to

clear solution with in 30 min. The solution can be stored for several months when refrigerated

at –20 oC.

The 5 l of stock sol–gel solution was vortexed with 45 l of phosphate buffer containing

0.5 U of enzyme stock solution. The 4 l of the enzyme sol was spread on the electrode

surface. This film was allowed to polymerize at room temperature for 3–5 min. This electrode

was gently washed with phosphate buffer (pH 7.0) and was used for further experimental

procedure [19]. The 0.04 U of enzyme was immobilization on the electrode surface.

3 . RESULTS AND DISCUSSION

3.1 . Cyclic voltammetric studies

The cyclic voltammogram (CV) of the sensor in the presence and absence of 1 mM

substrate in phosphate buffer (pH 7.0) at a scan rate of 10 mVs-1 is shown in Fig. 1.

In the absence of substrate, the fabricated biosensor working electrode doesn’t show any

response and only a small background current was observed in CV. When substrate was

added to the buffered solution anodic peak was observed at 0.60 V. The reaction mechanism

was shown in scheme.1. The investigated assay of AChE activity from the plot of current

versus acetylthiocholine chloride concentration has been shown in Fig. 2. The apparent

Michaelis–Menten constant (Kmapp) was thus estimated to be 500 M using the Lineweaver–

Burk plot of 1/I vs. 1/[acetylthiocholinechloride], where ‘I’ represents catalytic current of the

analyte. The relatively low Kmapp value indicates the high affinity of the enzyme towards the

substrate .

Anal. Bioanal. Electrochem., Vol. 4, No. 1, 2012, 1 – 16 6

Fig. 1. Cyclic voltammogram of enzyme electrode in 0.1 M phosphate buffer, pH 7.0 and

0.1 M KCl (a) without substrate (dashed line) (b) with 1 mM substrate (solid line)

Fig. 2. Calibration graph for acetylthiocholine chloride obtained with AChE immobilized

sensor in 0.1 M phosphate buffer / 0.1 M KCl at pH 7.0

Anal. Bioanal. Electrochem., Vol. 4, No. 1, 2012, 1 – 16 7

3.2. Effect of scan rate and pH

The effect of scan rate on the peak current at the enzyme modified carbon paste electrode

in 0.1 M KCl was investigated in the presence of 1 mM acetylthiocholine chloride. As shown

in the Fig. 3 the anodic peak current increases linearly with the square root of scan rate in the

range 5 to 50 mVs-1. The correlation coefficient was 0.9918, which indicate the electrode

reaction was diffusion controlled process [16].

Fig .3. The enzyme electrode response by varying square root of scan rate for 1 mM ASChCl

in 0.1 M phosphate buffer, pH 7.0 and 0.1 M KCl

The electrochemical response of enzyme modified carbon paste electrode towards 1 mM

acetylthiocholinechloride was generally pH dependent Fig. 4(A). As the pH of the solution

increases the response of the biosensor increases until attaining a physiological pH 7.0 and

from there onwards the response of the biosensor decreases. The anodic peak potentials of

acetylthiocholine chloride shifted from 0.68 to 0.51 V with respect to the pH from 5.5 to 8.5.

The potential diagram was constructed by plotting the graph of anodic peak potential Epa vs. pH of the solution. The enzyme electrode shows maximum sensitivity at pH 7.0 Fig. 4(B).

The pH dependence of oxidation peak potentials of substrate, Epa=0.9752+0.055pH

(r=0.9947).The graph has a good linearity with a slope of 55 mVs-1, this behavior nearly

obeys the Nernst equation for equal number of protons and electrons transfer reaction

[20, 21]. All experiments including inhibition studies were carried out at pH 7.0.

Anal. Bioanal. Electrochem., Vol. 4, No. 1, 2012, 1 – 16 8

3.3. Effect of enzyme concentration

The effect of enzyme concentration on peak current response with 1 mM substrate was

shown in Fig. 5. When the enzyme concentration is increased from 0.03 to 0.3 U in sol–gel

matrix entrapped onto the carbon paste electrode, it was noticed that peak current increases

up on increase in the enzyme concentration. The rate of enzyme catalyzed reaction was

dependent on the amount of enzyme immobilized. The highest sensitivity towards the

inhibitors was found with electrode containing low enzyme loading [22].

Fig. 4. (A) Effect of pH on the enzyme electrode response (B) Plot of Epa vs. pH to 1 mM

ASChCl

A

B

Anal. Bioanal. Electrochem., Vol. 4, No. 1, 2012, 1 – 16 9

Fig. 5. The effect of enzyme loading into sol–gel matrix on the response of biosensor system

3.4. Detection of organophosphate pesticides The biosensor was used for the detection of different organophosphorous pesticides such

as monocrotophos and phosphamidon. The sensor has been used to carry out inhibition

studies by incubation with pesticide solution to determine lower detection limits. To obtain an

inhibition plot the percentage inhibition method was followed. The electrode response was

first measured in 0.1 M phosphate buffer, 0.1 M KCl, pH 7.0 in presence of 1 mM

acetylthiocholine chloride this value corresponds to (Ii) the current before inhibition. The

electrode was washed with same buffer and incubated for 4 and 3 min with monocrotophos

and phosphamidon pesticide solution at a known concentration. The second value

corresponds to (IF), which was the current after inhibition. The inhibition percentage and

percentage of residual enzyme activity were calculated as follows [23].

Inhibition % (I %) = [(Ii–IF) / Ii] X 100 (1)

Residual enzyme activity % (REA %) = [IF / Ii] X 100 (2)

The organophosphorous compounds are known to inhibit the activity of AChE and in the

presence of monocrotophos and phosphamidon the rate of thiocholine production was

reduced. Quantitative analyses of individual pesticides were carried out according to the

above procedure. Calibration plots based on the dependence of the % inhibition on

concentration were linear and shown in Fig.6 A&B for monocrotophos and phosphamidon

respectively.

Anal. Bioanal. Electrochem., Vol. 4, No. 1, 2012, 1 – 16 10

Fig. 6. Inhibition plots of (A) Monocrotophos after 4 min incubation time (B) Phosphamidon

after 3 min incubation time in 0.1 M phosphate buffer at pH 7.0, 0.1 M KCl

The detection limit (DL) and quantification limit (QL) values were found to be 45 ppb,

0.06 ppm and 151 ppb, 0.2 ppm for monocrotophos and phosphamidon respectively. It was

seen that the lowest DL value was achieved with monocrotophos; this clearly indicates that

the monocrotophos was more toxic than phosphamidon. The behavior of enzyme activity

within the concentration range of 50–900 ppb and 0.1–1.25 ppm at different incubation times

were shown in Fig.7 A&B for monocrotophos and phosphamidon respectively.

A

B

Anal. Bioanal. Electrochem., Vol. 4, No. 1, 2012, 1 – 16 11

Fig. 7. Effect of incubation time in various inhibitor concentrations on the residual enzyme

activity in 0.1 M phosphate buffer / KCl, pH 7.0 (A) For monocrotophos (a) 50 ppb (b) 80

ppb (c) 100 ppb (d) 150 ppb (e) 300 ppb (f) 450 ppb (g) 800 ppb (h) 900 ppb(B) For

phosphamidon (a) 0.1 ppm (b) 0.3 ppm (c) 0.5 ppm (d) 0.7 ppm (e) 1.0 ppm (g) 1.25 ppm

It reveals that the level of inhibition of enzyme increases with increase in incubation time

and as well as increase in concentration of pesticides. A complete inhibition was observed at

shorter incubation times of 4&3 min for monocrotophos and phosphoamidon of

concentrations 900 ppb&1.25 ppm respectively. The inhibition effect of pesticides on the

immobilized enzyme was studied by employing differential pulse voltammetry (DPV) Fig. 8.

A

B

Anal. Bioanal. Electrochem., Vol. 4, No. 1, 2012, 1 – 16 12

When the concentration of pesticides increases the residual enzyme activity decreases with

respect to different time intervals. This was shown in Fig. 9A & B for monocrotophos and

phosphamidon. Determination of DL and QL was calculated by using the following

expression [16, 24–26].

DL = 3 Sb/S (3)

QL = 10 Sb/S (4)

Where ‘Sb’ is standard deviation of the mean values for ten voltammograms of the blank

solution, S is the slope of the working curve, DL is the detection limit, and QL is the

quantification limit. Table 2 shows the various parameters determined for monocrotophos and

phosphamidon.

Fig. 8. Differential pulse voltammogram of (a) substrate alone (b) with 900 ppb

monocrotophos/1.25 ppm phosphoamidon pesticide solutions

Anal. Bioanal. Electrochem., Vol. 4, No. 1, 2012, 1 – 16 13

Fig. 9. The variation of residual enzyme activity with different inhibitor concentrations with

time in 0.1 M phosphate buffer / KCl (A) Monocrotophos (B) Phosphamidon

A

B

Anal. Bioanal. Electrochem., Vol. 4, No. 1, 2012, 1 – 16 14

Table 2. The various parameters determined for monocrotophos and phosphamidon

Parameters Monocrotophos Phosphamidon

Response time (min) 1 1

Incubation time (min) 4 3

Linear range 50 – 900 ppb 0.1 – 1.25 ppm

Intercept of calibration curve 28.63 33.52

Slope of calibration curve 0.0772 0.0566

Correlation coefficient 0.9926 0.9671

Standard deviation (S.D.) 3.6703 7.1844

Detection limit (DL) 45 ppb 0.06 ppm

Quantification limit (QL) 151 ppb 0.2 ppm

4. CONCLUSIONS

The work describes a new biosensor for the determination of pesticides monocrotophos

and phosphamidon, which was developed and characterized. The enzyme electrode provided

good linearity to the pesticide concentration range. The AChE has been successfully

immobilized through a simple sol–gel technology, which was taking minimal preparation

time. The enzyme was well immobilized with in sol–gel matrix, which retained satisfactory

enzymatic catalytic activities. The advantage of this biosensor with AChE enzymes provided

a high sensitivity, ease of preparation, selectivity and lower detection limits for the analysis

of organophosphorous compounds. Moreover, the proposed procedure for enzyme

immobilization could be extended to other enzymes as it is simple, fast and very efficient. In

addition, the developed procedure can be employed for the monitoring of different types of

organic pollutants and thus enlarging the future applicability of the biosensor.

Acknowledgements

The authors are very thankful to the Department of Science and Technology (DST),

Government of India, New Delhi for the funding given through project no SR/FT/CS -

025/2009.

Anal. Bioanal. Electrochem., Vol. 4, No. 1, 2012, 1 – 16 15

REFERENCES

[1] M. V. Russo, L. Campanella, and P. Avino, J. Chromatogr. B 780 (2002) 431.

[2] I. Palchetti, A. Cagnini, M. Del Carlo, C. Coppi, M. Mascini, and A. P. F. Turner, Anal.

Chim. Acta 337 (1997) 315.

[3] Ni. Yongnian, Q. Ping, and S. Kokot, Anal. Chim. Acta 516 (2004) 7.

[4] S. S. E. Petropoulou, A. Tsarbopoulos, and P. A Siskos, Anal. Bioanal. Chem. 385

(2006) 1444.

[5] N. M. Brito, S. Navickiene, L. Polese, E. F. G. Jarelim, R. B. Abakerli, and M. L.

Ribeiro, Chromatrography A. 957 (2002) 201.

[6] A. G. S. Predo and C. Airoldi 57 (2001) 640.

[7] V. A. Pedrosa, S. A. S. Machado, and L. A. Avaca, Anal. Lett. 39 (2006) 1955.

[8] V. A. Pedrosa, L. Codognoto, and L. A. Avaca, J. braz. Chem. Soc. 14 (2003) 530.

[9] B. Prieto–Simon, M. Campus, S. Andreescu, and J. L. Marty, Sensors 6 (2006) 1161.

[10] X. H. Li, Z. H. Xie, H. Min, Y. Z. Xian, and L. T. Jin, Electroanalysis 19 (2007) 2551.

[11] S. Suwansa–Ard, P. Kanatharana, P. Asawatreratana kul, C. Limsakul, B.

Wongkittisuksa, and P. Thavarungkul, Biosens. Bioelectron. 21 (2005) 445.

[12] F. H. Diekey, Specific Adsorption, J. Phys. Chem. 59 (1955) 695.

[13] S. Braun, S. Rappoport, R. Zusman, D. Avnir, and M. Ottolenghi, Mater. Lett. 10

(1990) 1.

[14] L. M. Ellerby, C. R. Nishida, F. Nishida, S. A. Yamanaka, B. Dunn, J. S. Valentine, and

J. I. Zink, Science. 255 (1992) 1113.

[15] Y. Tatsu, K. Yamashitta, M. Yamaguchi, S. Yamamura, H. Yamamoto, and S.

Yoshikowa, Chem. Lett. (1992) 1615.

[16] U. Chandra, B. E. Kumara Swamy, O. Gilbert, and B. S. Sherigara, Electrochemica

Acta 55 (2010) 7166.

[17] O. Gilbert, B. E. Kumara Swamy, U. Chandra, and B. S. Sherigara, J. Electroanal.

Chem. 636 (2009) 80.

[18] S. Chithravathi, B. E. Kumara Swamy, U. Cahndra, G. P. Mamatha and B. S. Sherigara,

J. Electroanal. Chem. 645 (2010) 10.

[19] K. Anitha, S. V. Mohan, S. J. Reddy, Biosens. Bioelectron. 20 (2004) 848.

[20] B. D. Jones, and J. D. Ingle, Jr. Talanta 55 (2001) 699.

[21] M. C. Shen, H. C. Gheng, and V. S. Vasantha, J. Electroanal. Chem. 588 (2006) 235.

[22] A. A. Ciucu, C. Negelescu, and R. P. Baldwin, Biosen. Bioelectron. 18 (2003) 303.

[23] A. Kildebrandt, J. Ribas, R. Bragos, J. L Marty, M. T. Chez, and S. Lacorate, Talanta

75 (2008) 1208.

[24] T. M. Reddy and S. J. Reddy, Anal. Let. 37 (2004) 2079.

[25] T. M. Reddy, M. Sreedhar, S. J. Reddy, J. Pharm. Biomed. Anal. 31 (2003) 811.

Anal. Bioanal. Electrochem., Vol. 4, No. 1, 2012, 1 – 16 16

[26] R. N. Hegde, B. E. Kumara Swamy, N. P. Shetti, and S. T. Nandibewoor, J. Electroanal.

Chem. 635 (2009) 51.

Copyright © 2012 by CEE (Center of Excellence in Electrochemistry)

ANALYTICAL & BIOANALYTICAL ELECTROCHEMISTRY (http://www.abechem.com)

Reproduction is permitted for noncommercial purposes.