K. Gangadhara Reddy et al.

, IJSID, 2012, 2 (5), 457-465

ISSN:2249-5347

IJSID

International Journal of Science Innovations and Discoveries

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METHOD

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Available online through www.ijsidonline.info

VOLTAMMETRIC DETERMINATION OF ACEPHATE PESTICIDE BY LIQUID STATE LIPASE ENZYMATIC INHIBITION Department of Chemistry, S.V.U. College of Sciences, Sri Venkateswara University, Tirupati-517502, Andhra Pradesh, India K. Gangadhara Reddy, G. Madhavi*, P.Jeevan Jyothi, A.Vijaya Bhaskar Reddy

Received: 24-08-2012 Accepted: 13-10-2012

*Corresponding Author

ABSTRACT Acephate, an organo-phosphorus pesticide was studied by an electro analytical method. This study is based on the determination of Lipase enzyme inhibition activity by the p-Nitro phenol is determined by cyclic voltammetry at an anodic peak potential of +0.06V. The current response under the influence of enzyme concentration, substrate concentration, pH and time variation effects on the hydrolysis of p-Nitro phenyl Acetate substrate were studied. A linear calibration for the Acephate was obtained in the the optimized conditions by following the incubation time of 25 min. The detection limit concentration, 125U of enzyme, pH 7.0, 25 min of hydrolysis time and incubation time of The inhibition character of the liquid state lipase enzyme in the presence of

hydrolysis of p-Nitro phenyl acetate to p-Nitro phenol. The in situ generated electro active

concentration range of 100M to 900M with a correlation co-efficient of 0.9873 under

Address: Name: G. Madhavi Place: S.V. University, Tirupati. AP, India E-mail: gmchem01@gmail.com

was found as 159.77M of Acephate with optimized conditions of 750M substrate 25 min and the proposed method has a quantification limit of 532.57M. The developed liquid state lipase enzyme sensor takes less time for analysis, and no preconcentration extraction was needed for the study. methods. Key words: lipase enzyme, p-Nitro phenyl Acetate, p-Nitro phenol, Acephate, Volta metric

INTRODUCTION

INTRODUCTION

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K. Gangadhara Reddy et al., IJSID, 2012, 2 (5), 457-465 in modern agriculture due to their high insecticidal activity and relatively low resolution [1]. Organophosphorus (OPs) pesticide toxicity is based on the irreversibly inactivate acetyl cholinesterase (AChE), which is essential to nerve function in insects, for poisoning[2]. The applied Organo-phosphorus pesticides degrade rapidly by hydrolysis on exposure to sunlight, air, and hazard to health and the environment
[4].

humans, and many other animals. Organo-phosphorus pesticides affect this enzyme in varied ways, and thus in their potential soil, although small amounts can be detected in food and drinking water. When these small amounts of pesticide residues are reliable quantification of trace level of Organo-phosporous (Ops) compounds is important for monitoring their potential Liquid Chromatography-diode array detection[8] have been widely used for the determination of the organophosphorus costlier, requires more time and complex in nature. electrode by bi-enzymatic immobilized method

Most of the synthesized Carbamate and Organo-phosphorus (OPs) pesticide compounds are widely used as pesticides INTRODUCTION

present in water, food and animal feeds create a potential hazard due to their high mammalian toxicity [3]. Hence, a rapid and Performance Liquid Chromatography[6], Gas Chromatography-Mass spectrometry[7] and Liquid-Solid Extraction followed by pesticide compounds. These methods are highly sensitive, capable to determine a large number of compounds but they are enzyme acetylcholinesterase and coupled with simple detectors were developed recently[9-11]. The electrochemical sensor
[13],

Most of the traditional analytical techniques are Gas Chromatography[5], High-

based on acetylene black-chitosan composite film modified electrode [12], ferophthalocyanine chemically modified carbon paste biosensors are applicable for continuous monitoring of various types of organo-phosporous pesticide residues in and substrate. To overcome this problem Lipase enzyme is an alternative method for the determination of pesticide residues. direct electrochemical responsive substrate. But based on the chemical properties of lipase enzyme some of the lipase enzyme alcohol and acid, based on this; surface acoustic wave impedance sensor [15] and potentiometric biosensors [16] are developed. enzyme and pesticide effected lipase enzyme Reagents and solutions The development of mono enzymatic lipase enzyme biosensors are very few for pesticide determination because of lack of acetylcholinesterase enzyme modified carbon paste electrode
[14],

As a good alternative and with the improvement of being quick and consistent, biosensors based on the inhibition of

environmental samples. However the development of these acetylcholinesterase enzyme biosensors requires costlier enzyme biosensors are developed. Lipase enzyme shows an intrinsic capability to catalyze carboxylic ester bonds to the corresponding

and

electrochemical method for the determination of Acephate an organophosphorus pesticide. The developed method is purely based on the production of alcoholic compound (p-Nitrophenol) by p-Nitro phenyl Acetate substrate hydrolysis by free lipase MATERIALS AND METHODS

Based on the chemical properties of lipase enzyme we have developed an indirect liquid state lipase enzyme

(EC 3.1.1.3, type VII, 700/mg) p-Nitrophenyl Acetate, Acephate were purchased from Sigma-Aldrich Chemicals. 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, acetone (GR grade) were procured from Himedia chemicals. Phosphate Buffer 0.1M was prepared by using 0.1M disodium hydrogen phosphate and 0.1M Sodium dihydrogen phosphate. All Chemicals were of analytical grade and aqueous solutions were prepared with double distilled water. The enzyme stock solutions were preserved at -5C. International Journal of Science Innovations and Discoveries, Volume 2, Issue 5, September-October 2012

All chemicals were obtained from commercial sources and used without further purification. Lipase from C. Rugosa

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K. Gangadhara Reddy et al., IJSID, 2012, 2 (5), 457-465 Apparatus connection to a personal computer was used for electrochemical measurement and treating of data. A conventional three electrode cell was employed throughout the experiments, with a bare carbon paste electrode (homemade cavity of 3.0 mm electrode. All the experiments were carried out at room temperature. Preparation of bare carbon paste electrode diameter) as a working electrode, saturated calomel electrode (SCE) as a reference electrode and a platinum wire as a counter agate mortar to produce a homogenous carbon paste. The paste was packed into the cavity of homemade PVC (3 mm in at the end of the tube [17]. Cyclic Volta metric study of lipase enzyme RESULTS AND DISCUSSION Cyclic voltammetric experiments were performed with a model No: CHI610D Electrochemical work station with a

diameter) and then smoothed on a weighing paper. The electrical contact was provided by copper wire connected to the paste The electrochemical behavior of in situ generated p-Nitrophenol from the Lipase enzymatic hydrolysis of p-

The bare carbon paste electrode was prepared by hand mixing of 70% fine graphite powder and 30% silicon oil in an

Nitrophenyl Acetate was examined. Fig.1 shows a cyclic voltammogram of the p-Nitro phenol in the absence and presence of 125 Units of Lipase enzyme with 750substrate in 0.1M phosphate buffer (pH7.0) at a scan rate of 50 mV/s. In the absence of enzyme and the substrate (blank) the electrode gave no response and only a small back ground current was potentials of + 0.05V [peak c] is obtained.

observed [peak a]. In the presence of only 750of substrate a little back ground peak current was observed [peak b]. When the enzyme was added to the 750of substrate and after 25 min a relatively large anodic peak current at a

Fig. 1Cyclic voltammograms of in situ generated p-nitro phenol in 0.1M phosphate buffer, pH 7.0: a) substrate alone; shown in the given below equation (1) according to the literature report [18]. b) substrate in the presence of 125U of enzyme At 50 mV/Sec scan rate.

The p-Nitrophenol molecule readily undergoes electro oxidation. Primarily, it is oxidized to nitro phenoxy radical as

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K. Gangadhara Reddy et al., IJSID, 2012, 2 (5), 457-465 This radical intermediate subsequently undergoes polymerization leading to the formation of a non-sticky thin film

C6H4NO2OH

C6H4NO2O + H+ + e- ......................... (1)

on the electrode surface. For further studies the non-sticky polymeric film electrode surface is removed by physically smoothing against a tissue paper [18]. The anodic peak current was increased with increase of scan rate. The result of the graph obtained is with a good linearity (Fig.2), which indicates the electrode reaction as a diffusion controlled process [19].

Fig.2 The effect of scan rate on anodic peak current for p- Nitrophenol in 0.1M phosphate buffer The effect of time, pH on hydrolysis of p-Nitrophenyl Acetate of pH 7.0.

(0.1M) phosphate buffer with 750substrate. The time Vs effective hydrolysis of substrate product (Fig.3 A) showed a

gradual increase in the current from 5 min to 25 min of hydrolysis and the increase is continued till a plateau level is reached

The effect of time variation on p-Nitrophenyl Acetate hydrolysis by 125U of mobilized Lipase enzyme is studied in

and after that there is no significant increment in the hydrolysis of the substrate up to one hour. Based on this experiment, 25 hydrolysis of p-Nitrophenyl Acetate to p-Nitrophenol and acetic acid in the presence of Lipase enzyme is studied in 0.1M

minutes of hydrolysis time was chosen for the effective study of Lipase enzyme activity for pesticide study. The effect of pH on phosphate buffer with pH 6.0-8.0. The graph showed in Fig.3 B (--) a maximum hydrolysis of p-Nitrophenyl Acetate to pthe value reported in the literature [20]. The potential diagram was constructed by plotting the graph of anodic peak potential Nitro phenol reveals that there is a potential shift towards positive, and the Ep = 0.5752+0.071 this graph is almost linear transfer reaction [21-22]. Epa Vs pH of the solution and is shown in Fig.3 B (--). The pH dependence of oxidation peak potential of in situ generated pwith a slope of 71mV/ pH, this behavior was nearly obeying the Nernst equation for equal number of electron and proton Nitrophenol and acetic acid with liquid state lipase at pH 7.0 and also the obtained experimental value was almost similar to

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K. Gangadhara Reddy et al., IJSID, 2012, 2 (5), 457-465

Fig. 3 A. Time variation on enzymatic hydrolysis of substrate.

B. The effect of pH on hydrolysis of 750M substrate in 0.1M phosphate buffer (--)

The enzyme and substrate concentration effect on the hydrolysis of substrate

known that the selection of a proper enzyme and substrate concentration is important in enzyme inhibition studies. When the response and this response was finally saturated at 125U of enzyme concentration where the current values observed were observation 750M of substrate was taken as an optimum concentration of the substrate.

concentration of the enzyme is gradually increased from 25U to 250U there was a gradual increase in the peak current

The effect of enzyme and substrate concentration on the hydrolysis of p-Nitrophenyl Acetate was studied. It is well

B. The effect of pH on anodic peak potential (Epa) of p-Nitrophenol in 0.1M phosphate buffer (--)

almost constant (Fig.4 A) and optimized enzyme concentration was125U. When the substrate concentration was increased from 125M to 750M the p-Nitrophenol production was linearly increased and after that when the concentration of the

substrate was increased from 750M to 1750M the production of p-Nitrophenol was decreased (Fig. 4B). From the above

Fig.4 A. Response of increasing concentrations of Lipase enzyme in 0.1M phosphate buffer pH 7.0. International Journal of Science Innovations and Discoveries, Volume 2, Issue 5, September-October 2012 B. The effect of various substrate concentrations on enzymatic hydrolysis.

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K. Gangadhara Reddy et al., IJSID, 2012, 2 (5), 457-465 Pesticide study obtain the lower detection limits. Acephate was mixed in 1:1 ratio with 125U of enzyme stock solution and incubated at a room temperature of 252 C. To obtain an inhibition plot for Acephate pesticide the percentage of inhibition method was studied. The detection was based on the measurement of initial steady state current (Ii) response of complete hydrolysis of liquid state enzyme towards the selected concentration of 750 substrate in a 0.1M phosphate buffer solution at pH 7.0. inhibition (%) and residual enzyme activity was determined according to the following equations (2) and (3) [10, 23]. Inhibition % I(%)= [(Ii-If) / If] 100 ------------------------------------------------------------- (2) Residual enzyme activity % (REA %) = [ I f / Ii ] 100 ----------------------------------------- (3) The 1:1 ratio of enzyme and pesticide solution was incubated for 25 min, following the transfer of the inhibited enzyme into The Lipase enzyme was used to carry out inhibition studies by incubating with pesticide solution up to 25 min to

the electrochemical cell for the final steady state current (I f) response towards the hydrolysis of 750 substrate. The rate of Acephate pesticide is known to inhibit the catalytic nature of lipase enzyme towards the substrate, therefore toxic

reference to test the lipase enzyme activity was chosen for studies. Fig.5 shows various differential pulse voltammograms of

substrate alone, (Fig.5 peak c) indicating the presence of little amount of p-Nitrophenol, a starting substance for synthesis of pNitrophenyl Acetate substrate, (Fig.5 peak b) indicates enzyme inhibition in the presence of pesticide and the catalysis enzyme inhibition at various pesticide concentration levels. towards substrate inhibition was completely observed which is evidenced by the reduction in the current response. (Fig.5 Peak a) represents the complete hydrolysis of substrate in the presence of enzyme, the remaining voltammograms shows the

(Fig.6), the detection limit and the limit of quantification values were 159.77M, 532.57M respectively for Acephate. Various concentrations ranging from 100 to 900 M were tested in terms of their effect on enzyme activity at different incubation carried by using the following formulae (4) and (5) [24, 25]. times (5, 10, 15, 20 and 25 min) in pesticide solutions. When the concentration of Acephate increases the residual enzyme International Journal of Science Innovations and Discoveries, Volume 2, Issue 5, September-October 2012 activity of the enzyme decreases with time (Fig.7.). Determination of detection limit (DL) and quantification limit (QL) were

Calibration plots based on the dependence of the percentage of inhibition on concentration are linear and shown in

Fig.5 Differential pulse voltammograms of (c) substrate alone, (b) enzyme inhibition in the presence of pesticide, (a) complete hydrolysis of substrate in the presence of enzyme.

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quantification limit. Table.1 shows the various parameters determined for Acephate.

DL = 3Sb/S --------------------------------------------------------------- (4)

K. Gangadhara Reddy et al., IJSID, 2012, 2 (5), 457-465

QL = 10Sb/S -------------------------------------------------------------- (5)

Where Sb is standard deviation, S is the slope of the working curve, DL is the detection limit and QL is the

Fig.6 Inhibition plots of Acephate after 25 min incubation time with the measurement condition in 0.1M phosphate buffer.

Fig. 7 The effect of incubation time at various inhibitor concentrations on the activity of mobilized Lipase 450M, (e) 600 M, (f) 800 M, (g) 900 M enzyme in 0.1M phosphate buffer pH 7.0 for Acephate: (a) 100M, (b) 200 M, (c) 300M, (d)

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K. Gangadhara Reddy et al., IJSID, 2012, 2 (5), 457-465

Sl.No 1 2 3 4 5 6 7

Parameters Incubation time (min) Response time (min) Linear range (M ) Correlation coefficient Standard deviation Detection limit (DL) (M) Quantification limit (QL) (M)

Acephate 25 5 100-900 0.9873 4.21574 159.77 532.57

Table-1 Various parameters determined for Acephate pesticide.

The present study described the preparation of lipase based mobilized electrochemical sensor within the CONCLUSION

electrochemical cell to determine the concentration of an Acephate organo-phosphorus pesticide. This method is based on the enzyme inhibition method.

study of inhibition percentage of lipase enzyme activity. Electro analytical investigation of Acephate is achieved down to determination of Acephate in the environmental samples.

100M (correlation coefficient=0.9873 and slope=0.07905) at pH 7.0 and the plot obtained with a concentration of Acephate

Vs inhibition percentage yields a straight line almost passing through the origin rendering it suitable for electro analysis by The authors are thankful to the University Grants Commission, New Delhi, Government of India for providing the ACKNOWLEDGEMENT REFERENCES This developed method was simple, cost effective, eco-friendly and can be used for the

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