Voltametric determination of acephate pesticide by liquid state lipase enzymatic inhibition method


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Voltametric determination of acephate pesticide by liquid state lipase enzymatic inhibition method

  1. 1. K. Gangadhara Reddy et al., IJSID, 2012, 2 (5), 457-465 ISSN:2249-5347 IJSID International Journal of Science Innovations and Discoveries An International peer Review Journal for Science Research Article Available online through www.ijsidonline.info VOLTAMMETRIC DETERMINATION OF ACEPHATE PESTICIDE BY LIQUID STATE LIPASE ENZYMATIC INHIBITION METHOD 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 ReddyReceived: 24-08-2012 ABSTRACT The inhibition character of the liquid state lipase enzyme in the presence ofAccepted: 13-10-2012 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*Corresponding Author hydrolysis of p-Nitro phenyl acetate to p-Nitro phenol. The in situ generated electro active 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 concentration range of 100μM to 900μM with a correlation co-efficient of 0.9873 under the optimized conditions by following the incubation time of 25 min. The detection limit was found as 159.77M of Acephate with optimized conditions of 750μM substrate concentration, 125U of enzyme, pH 7.0, 25 min of hydrolysis time and incubation time of INTRODUCTION 25 min and the proposed method has a quantification limit of 532.57μM. The developedAddress: liquid state lipase enzyme sensor takes less time for analysis, and no preconcentrationName: G. Madhavi extraction was needed for the study. Key words: lipase enzyme, p-Nitro phenyl Acetate, p-Nitro phenol, Acephate, Volta metricPlace: S.V. University, Tirupati. AP, India methods. INTRODUCTIONE-mail: gmchem01@gmail.com International Journal of Science Innovations and Discoveries, Volume 2, Issue 5, September-October 2012 457
  2. 2. K. Gangadhara Reddy et al., IJSID, 2012, 2 (5), 457-465 Most of the synthesized Carbamate and Organo-phosphorus (OPs) pesticide compounds are widely used as pesticides INTRODUCTIONin modern agriculture due to their high insecticidal activity and relatively low resolution [1]. Organophosphorus (OPs) pesticidetoxicity is based on the irreversibly inactivate acetyl cholinesterase (AChE), which is essential to nerve function in insects,humans, and many other animals. Organo-phosphorus pesticides affect this enzyme in varied ways, and thus in their potentialfor poisoning[2]. The applied Organo-phosphorus pesticides degrade rapidly by hydrolysis on exposure to sunlight, air, andsoil, although small amounts can be detected in food and drinking water. When these small amounts of pesticide residues arepresent in water, food and animal feeds create a potential hazard due to their high mammalian toxicity [3]. Hence, a rapid andreliable quantification of trace level of Organo-phosporous (Ops) compounds is important for monitoring their potentialhazard to health and the environment [4]. Most of the traditional analytical techniques are Gas Chromatography[5], High-Performance Liquid Chromatography[6], Gas Chromatography-Mass spectrometry[7] and Liquid-Solid Extraction followed byLiquid Chromatography-diode array detection[8] have been widely used for the determination of the organophosphoruspesticide compounds. These methods are highly sensitive, capable to determine a large number of compounds but they arecostlier, requires more time and complex in nature. As a good alternative and with the improvement of being quick and consistent, biosensors based on the inhibition ofenzyme acetylcholinesterase and coupled with simple detectors were developed recently[9-11]. The electrochemical sensorbased on acetylene black-chitosan composite film modified electrode [12], ferophthalocyanine chemically modified carbon pasteelectrode by bi-enzymatic immobilized method [13], acetylcholinesterase enzyme modified carbon paste electrode [14], andbiosensors are applicable for continuous monitoring of various types of organo-phosporous pesticide residues inenvironmental samples. However the development of these acetylcholinesterase enzyme biosensors requires costlier enzymeand substrate. To overcome this problem Lipase enzyme is an alternative method for the determination of pesticide residues.The development of mono enzymatic lipase enzyme biosensors are very few for pesticide determination because of lack ofdirect electrochemical responsive substrate. But based on the chemical properties of lipase enzyme some of the lipase enzymebiosensors are developed. Lipase enzyme shows an intrinsic capability to catalyze carboxylic ester bonds to the correspondingalcohol and acid, based on this; surface acoustic wave impedance sensor [15] and potentiometric biosensors [16] are developed. Based on the chemical properties of lipase enzyme we have developed an indirect liquid state lipase enzymeelectrochemical method for the determination of Acephate an organophosphorus pesticide. The developed method is purelybased on the production of alcoholic compound (p-Nitrophenol) by p-Nitro phenyl Acetate substrate hydrolysis by free lipaseenzyme and pesticide effected lipase enzyme MATERIALS AND METHODS All chemicals were obtained from commercial sources and used without further purification. Lipase from C. RugosaReagents and solutions(EC, type VII, ≥ 700/mg) p-Nitrophenyl Acetate, Acephate were purchased from Sigma-Aldrich Chemicals. The pesticidestock solution was prepared by dissolving in acetone (GR grade solution). The graphite fine powder was procured from LoboChemie and Silicon oil, acetone (GR grade) were procured from Himedia chemicals. Phosphate Buffer 0.1M was prepared byusing 0.1M disodium hydrogen phosphate and 0.1M Sodium dihydrogen phosphate. All Chemicals were of analytical grade andaqueous solutions were prepared with double distilled water. The enzyme stock solutions were preserved at -5C. International Journal of Science Innovations and Discoveries, Volume 2, Issue 5, September-October 2012 458
  3. 3. K. Gangadhara Reddy et al., IJSID, 2012, 2 (5), 457-465 Cyclic voltammetric experiments were performed with a model No: CHI610D Electrochemical work station with aApparatusconnection to a personal computer was used for electrochemical measurement and treating of data. A conventional threeelectrode cell was employed throughout the experiments, with a bare carbon paste electrode (homemade cavity of 3.0 mmdiameter) as a working electrode, saturated calomel electrode (SCE) as a reference electrode and a platinum wire as a counterelectrode. All the experiments were carried out at room temperature. The bare carbon paste electrode was prepared by hand mixing of 70% fine graphite powder and 30% silicon oil in anPreparation of bare carbon paste electrodeagate mortar to produce a homogenous carbon paste. The paste was packed into the cavity of homemade PVC (3 mm indiameter) and then smoothed on a weighing paper. The electrical contact was provided by copper wire connected to the pasteat the end of the tube [17].Cyclic Volta metric study of lipase enzyme RESULTS AND DISCUSSION The electrochemical behavior of in situ generated p-Nitrophenol from the Lipase enzymatic hydrolysis of p- 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 750μsubstrate 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 observed [peak a]. In the presence of only 750μof substrate a little back ground peak current was observed [peak b]. When the enzyme was added to the 750μof substrate and after 25 min a relatively large anodic peak current at a potentials of + 0.05V [peak c] is obtained. Fig. 1Cyclic voltammograms of in situ generated p-nitro phenol in 0.1M phosphate buffer, pH 7.0: a) substrate alone; 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 shown in the given below equation (1) according to the literature report [18]. International Journal of Science Innovations and Discoveries, Volume 2, Issue 5, September-October 2012 459
  4. 4. K. Gangadhara Reddy et al., IJSID, 2012, 2 (5), 457-465 C6H4NO2O + H+ + e- ......................... (1) This radical intermediate subsequently undergoes polymerization leading to the formation of a non-sticky thin film C6H4NO2OHon the electrode surface. For further studies the non-sticky polymeric film electrode surface is removed by physicallysmoothing against a tissue paper [18]. The anodic peak current was increased with increase of scan rate. The result of the graphobtained 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 of pH 7.0. The effect of time variation on p-Nitrophenyl Acetate hydrolysis by 125U of mobilized Lipase enzyme is studied inThe effect of time, pH on hydrolysis of p-Nitrophenyl Acetate(0.1M) phosphate buffer with 750μsubstrate. The time Vs effective hydrolysis of substrate product (Fig.3 A) showed agradual increase in the current from 5 min to 25 min of hydrolysis and the increase is continued till a plateau level is reachedand after that there is no significant increment in the hydrolysis of the substrate up to one hour. Based on this experiment, 25minutes of hydrolysis time was chosen for the effective study of Lipase enzyme activity for pesticide study. The effect of pH onhydrolysis of p-Nitrophenyl Acetate to p-Nitrophenol and acetic acid in the presence of Lipase enzyme is studied in 0.1Mphosphate buffer with pH 6.0-8.0. The graph showed in Fig.3 B (-●-) a maximum hydrolysis of p-Nitrophenyl Acetate to p-Nitrophenol and acetic acid with liquid state lipase at pH 7.0 and also the obtained experimental value was almost similar tothe value reported in the literature [20]. The potential diagram was constructed by plotting the graph of anodic peak potentialEpa Vs pH of the solution and is shown in Fig.3 B (-■-). The pH dependence of oxidation peak potential of in situ generated p-Nitro phenol reveals that there is a potential shift towards positive, and the Ep = 0.5752+0.071 this graph is almost linearwith a slope of 71mV/ pH, this behavior was nearly obeying the Nernst equation for equal number of electron and protontransfer reaction [21-22]. International Journal of Science Innovations and Discoveries, Volume 2, Issue 5, September-October 2012 460
  5. 5. 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 750μM substrate in 0.1M phosphate buffer (-●-) B. The effect of pH on anodic peak potential (Epa) of p-Nitrophenol in 0.1M phosphate buffer (-■-) The effect of enzyme and substrate concentration on the hydrolysis of p-Nitrophenyl Acetate was studied. It is wellThe enzyme and substrate concentration effect on the hydrolysis of substrateknown that the selection of a proper enzyme and substrate concentration is important in enzyme inhibition studies. When theconcentration of the enzyme is gradually increased from 25U to 250U there was a gradual increase in the peak currentresponse and this response was finally saturated at 125U of enzyme concentration where the current values observed werealmost constant (Fig.4 A) and optimized enzyme concentration was125U. When the substrate concentration was increasedfrom 125μM to 750μM the p-Nitrophenol production was linearly increased and after that when the concentration of thesubstrate was increased from 750μM to 1750μM the production of p-Nitrophenol was decreased (Fig. 4B). From the aboveobservation 750μM of substrate was taken as an optimum concentration of the substrate. Fig.4 A. Response of increasing concentrations of Lipase enzyme in 0.1M phosphate buffer pH 7.0. B. The effect of various substrate concentrations on enzymatic hydrolysis. International Journal of Science Innovations and Discoveries, Volume 2, Issue 5, September-October 2012 461
  6. 6. K. Gangadhara Reddy et al., IJSID, 2012, 2 (5), 457-465 The Lipase enzyme was used to carry out inhibition studies by incubating with pesticide solution up to 25 min toPesticide studyobtain the lower detection limits. Acephate was mixed in 1:1 ratio with 125U of enzyme stock solution and incubated at aroom temperature of 25±2 °C. To obtain an inhibition plot for Acephate pesticide the percentage of inhibition method wasstudied. The detection was based on the measurement of initial steady state current (Ii) response of complete hydrolysis ofliquid state enzyme towards the selected concentration of 750 substrate in a 0.1M phosphate buffer solution at pH 7.0.The 1:1 ratio of enzyme and pesticide solution was incubated for 25 min, following the transfer of the inhibited enzyme intothe electrochemical cell for the final steady state current (I f) response towards the hydrolysis of 750 substrate. The rate ofinhibition (%) 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) Acephate pesticide is known to inhibit the catalytic nature of lipase enzyme towards the substrate, therefore toxicreference to test the lipase enzyme activity was chosen for studies. Fig.5 shows various differential pulse voltammograms ofsubstrate alone, (Fig.5 peak c) indicating the presence of little amount of p-Nitrophenol, a starting substance for synthesis of p-Nitrophenyl Acetate substrate, (Fig.5 peak b) indicates enzyme inhibition in the presence of pesticide and the catalysistowards substrate inhibition was completely observed which is evidenced by the reduction in the current response. (Fig.5Peak a) represents the complete hydrolysis of substrate in the presence of enzyme, the remaining voltammograms shows theenzyme inhibition at various pesticide concentration levels. 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. Calibration plots based on the dependence of the percentage of inhibition on concentration are linear and shown in(Fig.6), the detection limit and the limit of quantification values were 159.77μM, 532.57μM respectively for Acephate. Variousconcentrations ranging from 100 to 900 M were tested in terms of their effect on enzyme activity at different incubationtimes (5, 10, 15, 20 and 25 min) in pesticide solutions. When the concentration of Acephate increases the residual enzymeactivity of the enzyme decreases with time (Fig.7.). Determination of detection limit (DL) and quantification limit (QL) werecarried by using the following formulae (4) and (5) [24, 25]. International Journal of Science Innovations and Discoveries, Volume 2, Issue 5, September-October 2012 462
  7. 7. K. Gangadhara Reddy et al., IJSID, 2012, 2 (5), 457-465 DL = 3Sb/S --------------------------------------------------------------- (4) 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 thequantification limit. Table.1 shows the various parameters determined for Acephate. 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 enzyme in 0.1M phosphate buffer pH 7.0 for Acephate: (a) 100μM, (b) 200μM, (c) 300μM, (d) 450μM, (e) 600μM, (f) 800μM, (g) 900μM International Journal of Science Innovations and Discoveries, Volume 2, Issue 5, September-October 2012 463
  8. 8. K. Gangadhara Reddy et al., IJSID, 2012, 2 (5), 457-465 Sl.No Parameters Acephate 1 Incubation time (min) 25 2 Response time (min) 5 3 Linear range (μM ) 100-900 4 Correlation coefficient 0.9873 5 Standard deviation 4.21574 6 Detection limit (DL) (μM) 159.77 7 Quantification limit (QL) (μM) 532.57 Table-1 Various parameters determined for Acephate pesticide. The present study described the preparation of lipase based mobilized electrochemical sensor within the CONCLUSIONelectrochemical cell to determine the concentration of an Acephate organo-phosphorus pesticide. This method is based on thestudy of inhibition percentage of lipase enzyme activity. Electro analytical investigation of Acephate is achieved down to100μM (correlation coefficient=0.9873 and slope=0.07905) at pH 7.0 and the plot obtained with a concentration of AcephateVs inhibition percentage yields a straight line almost passing through the origin rendering it suitable for electro analysis byenzyme inhibition method. This developed method was simple, cost effective, eco-friendly and can be used for thedetermination of Acephate in the environmental samples. The authors are thankful to the University Grants Commission, New Delhi, Government of India for providing the ACKNOWLEDGEMENTfinancial assistance to carry out this work through major research project F. No.39-744/2010 (SR).1. R. Xue, T.F Kang, L.P Lu, S.Y Cheng; Applied Surface Science, 258, 6040– 6045 (2012). REFERENCES2. I.Marinov, Y.Ivanov, K. Gabrovska, T. Godjevargova; J. Mol. Catal. B-Enzym, 2, 67–75 (2010).3. N. Mionettol, J.L. Marty, I. Karube; Biosens. Bioelectron., 9, 463–470 (1994).4. R. Solna, S. Sapelnikova, P. Skladal, M.W. Nielsen, C. Carlsson, J. Emneus, T. Ruzgas; Talanta, 65, 349–357 (2005).5. A.D. Muccio, P. Pelosi, I. Camoni, D.A. Barbini, R. Dommarco, T. Generali, A. Ausil; J.Chromatogr., A 754, 497–506 (1996).6. P. Uutela, R. Reinila, P. Piepponen, R.A. Ketola, R. Kostiainen, Rapid Commun.Mass Spectrom., 19, 2950–2956 (2005).7. D. Srajnbahera, L.Z. Kraljb, J. Chromatogar A 1015 185-198 (2003).8. S. Lacorte, D. Barcelo, J. Chromatogar A 725 85-92 (1996).9. F.N. Kok, F. Bozoglu, V. Hasirci; Biosens. Bioeletron., 17, 531–539 (2002).10. K. Anitha, S.V. Mohan, S.J. Reddy, Biosens Bioelectron., 20, 848-856 (2004).11. B. Bucur, D. Fournier, A. Danet, J.-L. Marty, Anal. Chim. Acta, 562, 115–121 (2006).12. W. Yazhen, Q. Hongxin, H. Siqian, X. Junhui, Sensor Actuat B 147 587-592 (2010).13. A.A. Ciucu, C. Negulescu, R.P. Baldwin, Biosens Bioelectron 18 303-310 (2003). International Journal of Science Innovations and Discoveries, Volume 2, Issue 5, September-October 2012 464
  9. 9. K. Gangadhara Reddy et al., IJSID, 2012, 2 (5), 457-46514. D. D. Tuoro, M. Portaccio, M. Lepore, F. Arduini, D. Moscone, U. Bencivenga, D.G. Mita; New. Biotechnol 29 132-138 (2011).15. W. Wei, R. Wang, L. Nie, S. Yao; Instrum. Sci. Technol., 25, 157-167 (1997).16. F. Kartal, A. Kilinc, S. Timur, Intern. J. Environ. Anal. Chemistry, 87, 715-722 (2007).17. S. Reddy, B. E. Kumara Swamy, B.N. Chandrashekar, S. Chitravathi, H. Jayadevappa, Anal. Bioanal. Electrochem, 4, 186- 196 (2012).18. J. Mathiyarasu, J. Joseph, K.L.N. Phani, V. Yegnaraman, Indian. J. Chem. Techn., 11, 797-803 (2004).19. M.F.S. Teixeira, M.F. Bergamini, C.M.P. Marques, N. Bocchi; Talanta, 63, 1083-1088. (2004).20. G. Ozyilmaz, J. Mol. Catal. B-Enzym., 56, 231-236 (2009).21. S. Reddy, B.E.K. Swamy, H. Jayadevappa, Electrochim. Acta., 61, 78-86 (2012).22. O. Gilbert, B.E. Kumara Swamy, U. Chandra, B.S. Sherigara; J. Electroanal. Chem., 636, 80-85 (2009).23. A. Hildebrandt, J. Ribas, R. Bragos, J.L. Marty, M. Tresnchez, S. Lacorte; Talanta, 75, 1208-1213 (2008).24. R.N. Hegde, B.E. Kumara Swamy, N.P. Shetti, S.T. Nandibewoor; J. Electroanal. Chem., 635, 51-57 (2009).25. G. Madhavi, J. Damodar, S.K. Mohan, S.J. Reddy; B. Electrochem., 10, 209-213 (1998). International Journal of Science Innovations and Discoveries, Volume 2, Issue 5, September-October 2012 465