research article preparation of electrochemical biosensor...

Research ArticlePreparation of Electrochemical Biosensor for Detection ofOrganophosphorus Pesticides

Ashish Gothwal1 Puneet Beniwal1 Vikas Dhull2 and Vikas Hooda1

1Centre for Biotechnology Maharshi Dayanand University Rohtak 124001 India2Department of Bio- amp Nanotechnology Guru Jambeshwar University of Science amp Technology Hisar 125001 India

Correspondence should be addressed to Vikas Hooda vikascbtmdugmailcom

Received 8 September 2014 Accepted 10 December 2014 Published 24 December 2014

Academic Editor Teizo Kitagawa

Copyright copy 2014 Ashish Gothwal et alThis is an open access article distributed under theCreative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Polyvinyl chloride (PVC) can be used to develop reaction beaker which acts as electrochemical cell for the measurement of OPpesticides Being chemically inert corrosion resistant and easy in molding to various shapes and size PVC can be used for theimmobilization of enzyme Organophosphorus hydrolase was immobilized covalently onto the chemically activated inner surfaceof PVC beaker by using glutaraldehyde as a coupling agent The carbon nanotubes paste working electrode was constructedfor amperometric measurement at a potential of +08V The biosensor showed optimum response at pH 80 with incubationtemperature of 40∘C119870

119898

and 119868max for substrate (methyl parathion)were 32258120583Mand 11120583A respectively Evaluation study showeda correlation of 0985 which was in agreement with the standard method The OPH biosensor lost 50 of its initial activity afterits regular use for 25 times over a period of 50 days when stored in 01M sodium phosphate buffer pH 80 at 4∘C No interferencewas observed by interfering species

1 Introduction

Organophosphate (OP) compounds are used as pesticidesand insecticides in agriculture and have found applica-tions in military practices as chemical warfare agents OPcompounds contribute about 38 of the total pesticidesused worldwide [1] Commonly used OP pesticides includeparathion malathion methyl parathion chlorpyrifos diazi-non dichlorvos phosmet and fenitrothion According toWorld Health Organization reports there are three millionpesticide poisonings that occurred worldwide every yearmost of them are OP-related and 200000 deaths occur dueto either self-poisoning or occupational exposure [2] Reportsin the literature demonstrate that OP pesticides affect thenontarget organisms such as birds and fish as well as humansthrough exposure to water resources fruits vegetables andprocessed foods contaminated by OP pesticides The OPpesticides irreversibly inhibit the activity of acetylcholineesterase an enzyme essential for the proper functioning ofthe central nervous system in humans and insects resultingin the accumulation of the neurotransmitter acetylcholine inthe nerve cells and interfere with muscular responses and

cause serious problems and sometimes fatal [3ndash5] Analyticalmethods for determination of these toxic compounds arerequired to ensure that environment and human health willnot be compromised by the usage of OP pesticides Thereare many laboratory based analytical methods which arecommonly used for the determination ofOPpesticidesTheseinclude gas chromatography (GC) high-performance liquidchromatography (HPLC) capillary electrophoresis [6 7]mass spectrometry [8] and thermospray-mass spectrometry[9] Besides the sensitivity and accuracy of these methodsthey are expensive and time-consuming and require pre-treatment of the sample as well as requiring highly trainedpersons to perform them and are not suitable for in-fieldanalysis [8 10 11]

The need for rapid and cost-effective analytical methodsfor field analysis of OP pesticides has developed manytechniques in which few of them are bioanalytical in natureThese techniques are based on the inhibition assay [12] andimmunoassay [13] Enzyme-linked immunosorbent assay(ELISA) is quite sensitive assay for OP pesticides but likeother immunoassays can detect only single compound andrequire multiple time-consuming steps Inhibition assays

Hindawi Publishing CorporationInternational Journal of Analytical ChemistryVolume 2014 Article ID 303641 8 pageshttpdxdoiorg1011552014303641

2 International Journal of Analytical Chemistry

based on cholinesterase activity are also used for screeningprocesses But these methods are not well suited for processcontrol monitoring assays where fast and repetitive analysesare required [14]

These problems can be solved by the use of biosensortechnology which fulfils the demands for on-site monitoringand the rapid detection of neurotoxic agents [15] Biosensorsbased on the acetylcholine esterase (AChE) inhibition havebeen widely used for the determination of OP pesticides [16]The main drawback of inhibition-based biosensors involvesmultiple-step operation such as time-consuming incubationand reactivationregeneration steps [14 17]

Organophosphorus hydrolase (OPH) can catalyze thehydrolysis of a large number of OP pesticides [18] Theenzymatic hydrolysis of OP pesticides involves a pH changeas well as the generation of chromophoric species [19] OPH-based assays are direct and respond to OP pesticides assubstrate rather than inhibitors in case of inhibition-basedbiosensors Consequently these assays can be reversible andrequire only target analyte [14]Themain problem associatedwith these methods involves the use of free enzyme whichcan be used once and also increases the cost of procedureTo overcome this problem use of immobilizationmethod forconfining the enzyme molecules onto the different insolublesupports this enhances the reusability of enzyme throughimmobilization

Although OPH-based methods have several advantagessuch as selectivity for target analyte and show fast responsethey are less sensitive and have low detection limits ascompared to inhibition-based methods [20 21]

These problems can be overcome by using the carbonnanotubes (CNTs) as transducer material in amperometricbiosensors CNT showed excellent electrocatalytic propertieswith larger surface area [22] Deo et al which significantlyenhanced the amperometric signals generated from OPpesticides [23] The OPH biosensors provide a simple fastaccurate and selective monitoring of OP pesticides as wellas being good for in-field applications and monitoring ofdetoxification processes [19]

In the present method the OPH was extracted andpurified from Brevundimonas diminuta cells and used forthe development of electrochemical biosensor by covalentimmobilization of enzyme on to polyvinyl chloride (PVC)surface rather than working electrode This facilitates theopportunity to change the working electrode on deteriora-tion without wasting the immobilized enzyme which makesit economical and convenient The use of carbon nanotubesfor the construction of working electrode provides efficientcharge transfer from one phase to another The use ofpolyvinyl chloride (PVC) in bioanalytical techniques willopen a new pathway in biosensor design and fabricationdiagnostic kits and related biomedical instrumentation

2 Materials and Methods

21 Chemicals and Reagents Sephadex G-100 was purchasedfrom MP Biomedicals Nutrient broth tris-HCL EDTAlysozyme sucrose and sodium hydroxide were purchased

from Himedia Glutaraldehyde (grade 125) was purchasedfrom Sigma chemicals and organophosphorus (OP) pesti-cide (methyl parathion) was purchased from Shri Ram Fer-tilizers amp Chemicals All other chemicals were of analyticalreagent grade unless otherwise stated Double distilled water(DDW) was used throughout the experiments Silver wireand PVC (polyvinyl chloride) beaker were bought from localmarket

22 Instruments and Equipment Cyclic voltammetry (CV)measurements were performed on a potentiostatgalvanostat(PGSTAT1230302 Autolab) with a three-electrode systemcomposed of a platinum (Pt) wire as an auxiliary electrodean AgAgCl electrode as reference electrode and carbonnanotubes paste based working electrode UV spectropho-tometer (Shimadzu Corporation Japan) temperature con-trolled water bath (SunRon) digital pH meter (EUTECH)refrigerator (LG) microwave oven (LG) rotatory incubatorshaker (HICON) magnetic stirrer (HICON) refrigeratedcentrifuge (SIGMA) and deep freezer (Voltas) The study ofsurface morphology was carried out with scanning electronmicroscope (SEM) (Model Joel JSM-6510 Japan) at Depart-ment of Chemistry M D University Rohtak India

23 Microorganism The lyophilized sample of bacteria Bre-vundimonas diminuta (MTCC 3361) was purchased fromIMTECH Chandigarh

24 Revival and Growth of Bacteria Lyophilized sample ofB diminuta was mixed with 5mL of nutrient broth (pH-80)in a test tube to make a bacterial suspension The conicalflask (250mL) containing 100mL nutrient broth (pH-80)was inoculated with bacterial suspension The culture mediawere incubated in a rotatory incubator shaker at 200 rpm for24 hours at 30∘C Growth of bacteria was checked by takingOD at 600 nm

25 Extraction and Purification of OPH from Brevundimonasdiminuta After desirable growth the cells were centrifugedat 8000 rpm for 10 minutes The cell pellets was incubatedwith 075M Sucrose mixed in 75mL of 50mM SodiumPhosphate Buffer pH8 at 20∘C for 10minutesThe suspensionwar treated with lysozyme as described by Herzliger et al1984 [24] the suspension was centrifuged at 10000 rpm for 5minutesThe pallet was separated and re-suspended in 20mLof 50mM sodiumphosphate buffer (pH 8) containing 50mMNaCl at 4∘C for one hour with constant steering [25] Aftercentrifugation at 10000 rpm for 5 minutes the supernatantwas separated and treated as crud enzyme solutionThe crudesolution was concentrated using lyophilization machinePurification of concentrated crude Organophosphate hydro-lase was done using sephadex G-100 column (25 times 30 cm) asdescribed by Liu et al 2004 [26]

26 Enzyme Assay and Protein Estimation The 01mL ofpurified Organophosphate hydrolase enzyme solution wasmixed with 29mL Sodium Phosphate Buffer (pH 8) 5 nMmethyl parathion (01mL) was added to the enzyme solution

International Journal of Analytical Chemistry 3

Figure 1 Carbon nanotubes paste working electrode

and incubated at room temperature for 10 minutesThe spec-trophotometric reading of the assay was taken at 410 nm [27]The total protein content of the purified Organophosphatehydrolase enzyme solution was measured by Lowery method[28]

OP pesticide +H2O OPH997888997888997888rarr 4-nitophenol + diethyl phosphate

(120582 = 410 nm)(1)

27 Covalent Immobilization of OPH onto Inner Surface ofPVC Beaker Covalent immobilization of organophosphorushydrolase onto PVC beaker surface was done by using themethod described by Hooda et al [29] Inner surface ofPVC beaker was filled with 5mL of enzyme solution andkept overnight at 4∘C The surface was washed with buffer toremove the unbound enzymeThe reaction beaker was storedat 4∘C containing sodium phosphate buffer (50mM pH-80)

28 Scanning ElectronMicroscopy of PVCSurface To confirmthe immobilization of enzyme the scanning electron micro-scopic (SEM) study of the surface of PVC beaker was carriedout before and after enzyme immobilization at Departmentof Chemistry M D University Rohtak India

29 Preparation of Carbon Nanotubes Paste Working Elec-trode Carbon nanotubes powder (10 g) andNH

4Cl (02mg)

were mixed with paraffin oil in a ratio to obtain the consis-tency of paste [29] This paste was filled in a plastic hollowtube (2 cm length and 4mm diameter) with one closed endElectrical contact was made by inserting a silver wire into thecarbon nanotubes paste at one end (Figure 1)This formed thebody of the working electrode The surface of the electrodewas washed with buffer and stored at 4∘C when not in use

210 Assembly of OPH Biosensor and Electroanalytical Mea-surements An amperometric OPH biosensor was developed

AgAgCl electrode

Pt

CNTs based working electrode

PVC beaker

OPH immobilized in beaker

Figure 2 Basic assembly of OPH biosensor

by using enzyme immobilized PVC reaction beaker alongwith the three electrodes that is carbon nanotubes pasteas working electrode AgAgCl electrode as reference andPt wire as auxiliary electrode and being connected throughpotentiostatelectrochemical analyzer (Figure 2)

The electroanalytical measurements for OP pesticideswere performed using a potentiostatelectrochemical ana-lyzer The cyclic voltammetric study of 4-nitrophenol wasdone in 50mM sodium phosphate buffer (pH-80) in areaction beaker cell at room temperature In the cyclicvoltammetry experiments the scan rate was 50mVsec andthe scan range was 0V to +10 V

211 Kinetic Properties of Immobilized OPH-Based BiosensorThe following kinetic properties of OPH biosensor werestudied optimum pH incubation temperature time of incu-bation effect of substrate (methyl parathion) concentrationand calculation of 119870

119898and 119868max from Line weaver - Burk

plot between reciprocal of substrate concentration (1[119878]) andreciprocal of amount of the current (1[119868])

212 Evaluation of the Present Method The evaluation ofpresent method was carried out by studying its linearityminimum detection limit analytical recovery precision andaccuracy The effect of interfering species on the response ofbiosensor was studiedThe storage stability and reusability ofpresent method were also determined

2121 Linear Working Range and Minimum Detection LimitThe linearity and minimum detection limit were calculatedby correlating the values with standard graph

2122 Analytical Recovery To determine the reliability of themethod different concentrations of methyl parathion (5 and10 120583M) were added to the samples and the mean analyticalrecovery was determined by the present method

2123 Precision To study the reproducibility of the presentmethod the OP pesticide level was determined in the sampleon the same day (within batch) and in the same sample afterstorage at 4∘C for one week (between batch) coefficients ofvariation (CVs) were calculated for the present method

2124 Accuracy To evaluate the accuracy of presentmethodthe levels of methyl parathion in 5 spiked water samples were


determined by the present method and compared with thoseobtained by standard method

2125 Reusability and Storage Stability of the Present MethodBefore reuse of the OPH biosensor it was washed by washingbuffer (001M phosphate buffer saline pH-72 with 01tween 20) The storage stability of the present biosensor wasinvestigated over a period of two months when reactionbeaker was stored at 4∘CThe response of presentmethodwasmeasured once in every 5 days

2126 Effect of Interfering Species The amperometricresponse was also checked in the presence of potentialinterfering species such as glucose fructose and sucrose aswell as metal ions (Zn (II) Cu (II) Cd (II) Ni (II) and Pb(II)) each at a concentration of 2120583M

213 Application of Newly Developed OPH Biosensor Thewater and food samples prone to be contaminated with OPpesticides determination were taken for studyWater sampleswhich are used for the drinking purpose were used directlyfor the determination of OP pesticides The food samplesthat is vegetables and fruits (cauliflower cabbage grapesand apple) were bought from local market The food sampleswere washed with distilled water and chopped 100 gramsof each chopped sample was crushed in pestle mortar andhomogenize with 50mL of PBS (pH 70) and stirred for 1 hat room temperatureThe samples were filtered through filterpaper and centrifuged at 8000 rpm for 8min Supernatantwas collected and take 20mL of supernatant for OP pesticidemeasurements

OP compounds present in the sample were hydrolyzedto 4-nitrophenol by the OPH enzyme immobilized ontoPVC surface Optimum potential was applied to oxidize 4-nitrophenol and the response current generated was directlyproportional to the OP compound concentration in the sam-pleThis electrochemical technique is employed to determineOP pesticide concentration in food and water samples

3 Results and Discussion

31 Purification of Organophosphorus Hydrolase from Bre-vundimonas diminuta Enzyme was purified from crudeextract by gel filtration on Sephadex G-100 and ion exchangechromatography on DEAE-sepharoseThe enzyme was puri-fied by 1415-fold to a specific activity of 2081 Umg of proteinfrom the crudeOPH solutionwith a yield of 1992Thepuri-fied enzyme showed a single band at 36KDa on SDS-PAGE

32 Covalent Immobilization of OPH onto PVC SurfaceCovalent immobilization of enzyme on PVC beaker surfacewas carried out by method described previously [29] Innersurface of PVC beaker (30mL) capacity was treated with2mL of fuming nitric acid at 30∘C for about 2 h Afterthat PVC beaker was washed first with running tap waterand then with double distilled water The surface was thentreated with 1mL of methyl cyanide for 1 h and thereafterwith concentrated HCl (1mL) for 2 h The activated surface

of PVC was rinsed with distilled water and incubated with125 (wv) glutaraldehyde for about 8 h at 30∘CThe surfacewas then washed with distilled water to remove unboundglutaraldehyde Finally PVC beaker was poured with 5mLof enzyme solution containing 0245mg of OPH and keptovernight at 4∘C Then the surface was washed with bufferto remove the unbound enzyme

33 Scanning Electron Microscopy of PVC Surface The mor-phology of PVC beaker surface before and after the immo-bilization of enzyme was studied by SEM analysis Thechange on the chemically activated PVC surface (Figure 3)was observed after immobilization method Such a change insurface morphology of the PVC surface after immobilizationmethod confirms the enzyme immobilization

34 Determination ofWorking Potential of the OPH BiosensorThe amperometric determination of OP pesticides by OPH-based biosensor depends on the anodic detection of 4-nitrophenol (enzymatically liberated product) by the elec-trode [19] The working electrode showed a well-definedoxidation peak at a potential of +08V (Figure 4) Therefore+08V was selected as the working potential for the furtherstudies

35 Kinetic Study of the OPH Biosensor

351 Effect of pH The effect of pH on the biosensor responsein 01M succinate buffer (pH 60 65 and 70) sodiumphosphate buffer (75 80 and 85) and borate buffer (pH 9095 and 100) in 60ndash100 range was studiedThe optimum pHfor present method was 80 (Figure 5) which is higher thanpH-74 [19 23 30 31] pH 70 [32 33] and lower than pH 88[34] and pH 95 [17] This pH is similar to those reported inprevious study [35]

352 Effect of Incubation Temperature The effect of thetemperature on the biosensor response from 20∘C to 60∘C ata regular increase of 5∘C was also investigated The optimumtemperature for present method was 40∘C (Figure 6) Themicroenvironment provided by support used for immobiliza-tion makes it thermally stable and maintains its biologicalactivity

353 Effect of Time of Incubation Time of incubation wasalso studied from 5min to 20min at a regular increase of5min The response of the OPH biosensor was increased upto 10min after which no increase in response was observedThe optimum incubation time for presentmethodwas 10min(Figure 7)

354 Effect of Substrate Concentration The effect of methylparathion concentration on the response of present biosensorwas studied up to 500 120583M at an interval of 50 120583M Thepresent method showed a hyperbolic relationship betweenits response and methyl parathion concentration up to afinal concentration of 400120583M (Figure 8) after which nosignificant improvement in response was observed Up to a


(a)

(a)

(b)

(b)

Figure 3 Scanning electron micrographs of a chemically modified PVC surface without (a) and with (b) immobilized enzyme

350

300

250

200

150

100

50

0

0 02 04 06 08 1 12 14

Curr

ent (120583

A)

Potential (V)

Figure 4 Working electrode showing oxidation peak

12

1

08

06

04

02

0

6 65 7 75 8 85 9 95 10

pH

Curr

ent (120583

A)

Figure 5 Effect of pH on the response of OPH biosensor

concentration of 400120583M the OPH biosensor showed a goodresponse to methyl parathion

355 119870119898and 119868119898119886119909

119870119898and 119868max were determined by calcu-

lating the slope and intercept for the reciprocal plot of currentversus methyl parathion concentrations that is Lineweaver-Burk plot Lineweaver-Burk plot between the reciprocals ofmethyl parathion concentration and response current forbiosensor was linear119870

119898and 119868max were 32258 120583M and 11 120583A

(Figure 9)

12

1

08

06

04

02

0

Curr

ent (120583

A)

20 25 30 35 40 45 50 55 60

Temperature (∘C)

Figure 6 Effect of incubation temperature on the response of OPHbiosensor

0 5 10 15 20

Time of incubation (min)

12

1

08

06

04

02

0

Curr

ent (120583

A)

Figure 7 Effect of incubation time on the response of OPHbiosensor

12

1

08

06

04

02

0

Curr

ent (120583

A)

0 50 100 150 200 250 300 350 400 450 500

Substrate concentration (120583M)

Figure 8 Effect of methyl parathion concentration on the presentmethod


25

20

15

10

5

0

minus5minus002 0 002 004 006 008 01 012

1[I]

1[S]

Figure 9 Lineweaver-Burk plot of the present method

Table 1 Analytical recovery of added methyl parathion in sample

Methyl parathionadded (120583M)

Methyl parathionfound (120583M)

mean (119899 = 6) plusmn SD recovery

5 493 plusmn 002 98610 991 plusmn 002 991

Table 2 Within and between assay coefficients of variation fordetermination of OP pesticide in the samples by using the presentmethod

119899OP pesticide (120583M)

mean plusmn SD CV ()

Within batch (6) 184 plusmn 002 158Between batch (6) 183 plusmn 003 178

36 Evaluation of the Biosensor

361 Linear Working Range and Minimum Detection LimitThe linear working range and minimum detection limitof the present biosensor were calculated from standardgraph between response current and substrate concentration(methyl parathion) The linear range for methyl parathionwas found to be 01ndash200 120583M

362 Analytical Recovery The reliability of the presentmethod was determined by the analytical recovery of addedmethyl parathion (Table 1) The mean analytical recovery ofadded methyl parathion (5 120583M and 10 120583M) was 986 and991 respectively

363 Precision To study reproducibility of the presentmethod the OP pesticide level was determined in the samplerepeatedly on the same day (within batch) and in the samesample after storage at 4∘C for one week (between batch)The results of within batch and between batch coefficients ofvariation (CVs) were lt158 and lt178 (Table 2)

364 Accuracy To study the accuracy of the presentmethodthe levels of methyl parathion in 5 spiked water samples weredetermined by the standard method (119909) and by the presentmethod (119910) (Figure 10) The result obtained by the presentmethod was in agreement with the standard method with acorrelation of 0985

Table 3 Effect of various interfering species on response of thepresent method

Compound added (final conc 2120583M) relative responseNone 100Fructose 995Glucose 102Sucrose 994Zn(II) 983Cu(II) 987Cd(II) 992Ni(II) 973Pb(II) 977

30

25

20

15

10

5

00 5 10 15 20 25 30M

ethy

l par

athi

on co

ncen

trat

ion

(120583M

)in

spik

ed w

ater

by

pres

ent m

etho

d

Methyl parathion concentration (120583M)in spiked water by HPLC method

R2= 0985

Figure 10 Correlation between values of methyl parathion spikedwater samples determined by standard HPLC method (119909-axis) andby the present method (119910-axis)

12

1

08

06

04

02

0

Curr

ent (120583

A)

1 5 10 15 20 25

Number of uses

Figure 11 Reusability of the OPH biosensor

37 Reusability and Storage Stability of OPH Biosensor Thepresent biosensor lost 50of its initial activity after its 25 usesover a period of 50 days (Figures 11 and 12) when stored at4∘C which is better than previously reported biosensors [1733]

38 Interference Study Among the various substances inves-tigated for possible interference on the response of the presentmethod none caused any significant interference (Table 3)


12

1

08

06

04

02

0

Curr

ent (120583

A)

0 10 20 30 40 50

Storage stability (days)

Figure 12 Storage stability of the OPH biosensor

Table 4 OP pesticide concentration in different food samples

Serial number SampleConcentration of OPpesticide (mgkg)

(119899 = 3)1 Grape 01462 Apple 02433 Cauliflower 00314 Cabbage 0017119899 number of assays

Table 5 OP pesticide concentration in different water samples

Serial number SampleConcentration of OPpesticide (mglit)

(119899 = 3)1 Pond water 000912 Canal water 000373 Bore-well water 00029119899 number of assays

39 Application of Present Method in Determination of OPPesticides in Food and Water Samples The OP pesticidesin differed samples were determined by the newly devel-oped biosensor OP pesticide level in fruits and vegetableswas found in the range of 0146mgkgndash0243mgkg and0017mgkgndash0031mgkg respectively (Table 4)TheOP pes-ticide content of water samples was also determined andfound in the range 00029ndash00091mglit (Table 5)

4 Conclusion

The use of carbon nanotubes as electrode material facilitatesthe enhanced amperometric determination of OP pesticidesThe carbon nanotubes based transducer provides sensitiveand stable detection of the 4-nitrophenol (enzymaticallyliberated product of OP pesticides) The use of OPH forbiorecognition element and carbon nanotubes electrodefor amperometric determination provides advantages overAChE-based biosensors that are less specific towards OPpesticides and requires multiple steps PVC has emerged asan ideal support for enzyme immobilization and its use in

designing of present electrochemical cell has proved it betterthan other reported biosensorsThe present method providesa laboratory based analytical method for the determinationof OP pesticides with high specificity It provides fast deter-mination of OP pesticides as well as in-field monitoring Thepresent method can act as a model for the development ofindigenous OP pesticide sensor in miniature form

Conflict of Interests

Authors have no conflict of interests in publishing of thisresearch article

Acknowledgment

The authors acknowledge the financial support provided byUniversity Grants Commission New Delhi to Biosensorsand Diagnostics Laboratory Maharshi Dayanand UniversityRohtak India

References

[1] B K Singh ldquoOrganophosphorus-degrading bacteria ecologyand industrial applicationsrdquo Nature Reviews Microbiology vol7 no 2 pp 156ndash164 2009

[2] S B Bird T D Sutherland C Gresham J Oakeshott CScott andM Eddleston ldquoOpdA a bacterial organophosphorushydrolase prevents lethality in rats after poisoning with highlytoxic organophosphorus pesticidesrdquo Toxicology vol 247 no 2-3 pp 88ndash92 2008

[3] W J Donarski D P Dumas D P Heitmeyer V E Lewis and FM Raushel ldquoStructure-activity relationships in the hydrolysisof substrates by the phosphotriesterase from Pseudomonasdiminutardquo Biochemistry vol 28 no 11 pp 4650ndash4655 1989

[4] S Chapalamadugu and G R Chaudhry ldquoMicrobiological andbiotechnological aspects of metabolism of carbamates andorganophosphatesrdquo Critical Reviews in Biotechnology vol 12no 5-6 pp 357ndash389 1992

[5] J A ComptonMilitary Chemical and Biological Agents TelfordPress New Jersey NJ USA 1988

[6] J Sherma ldquoPesticidesrdquo Analytical Chemistry vol 65 no 12 pp40ndash54 1993

[7] S Yao A Meyer and G Henze ldquoComparison of amperometricand UV-spectrophotometric monitoring in the HPLC analysisof pesticidesrdquo Freseniusrsquo Journal of Analytical Chemistry vol339 no 4 pp 207ndash211 1991

[8] S Lacorte and D Barcelo ldquoRapid degradation of fenitrothionin estuarine watersrdquo Environmental Science and Technology vol28 no 6 pp 1159ndash1163 1994

[9] S Lacorte and D Barcelo ldquoDetermination of organophos-phorus pesticides and their transformation products in riverwaters by automated on-line solid-phase extraction followedby thermospray liquid chromatography-mass spectrometryrdquoJournal of Chromatography A vol 712 no 1 pp 103ndash112 1995

[10] I Karube K Yano S Sasaki Y Nomura and K IkebukuroldquoBiosensors for environmental monitoringrdquo Annals of the NewYork Academy of Sciences vol 864 pp 23ndash36 1998

[11] J Diel-Faxon A L Gjindilis P Atanasov and E WilkinsldquoFlow injection amperometric enzyme biosensor for direct


determination of organophosphate nerve agentsrdquo Sensors andActuators B vol 35 pp 448ndash457 1996

[12] P Skladal ldquoBiosensors based on cholinesterase for detection ofpesticidesrdquo Food Technology and Biotechnology vol 34 no 1 pp43ndash49 1996

[13] E P Meulenberg W H Mulder and P G Stoks ldquoImmunoas-says for pesticidesrdquo Environmental Science and Technology vol29 no 3 pp 553ndash561 1995

[14] A Mulchandani W Chen P Mulchandani J Wang and KR Rogers ldquoBiosensors for direct determination of organophos-phate pesticidesrdquo Biosensors and Bioelectronics vol 16 no 4-5pp 225ndash230 2001

[15] K R Rogers and C L Gerlach ldquoEnvironmental biosensors astatus reportrdquo Environmental Science and Technology vol 30pp 486Andash491A 1996

[16] I Palchetti A Cagnini M del Carlo C Coppi MMascini andA P F Turner ldquoDetermination of anticholinesterase pesticidesin real samples using a disposable biosensorrdquoAnalytica ChimicaActa vol 337 no 3 pp 315ndash321 1997

[17] PMulchandaniW Chen and AMulchandani ldquoFlow injectionamperometric enzyme biosensor for direct determination oforganophosphate nerve agentsrdquo Environmental Science andTechnology vol 35 no 12 pp 2562ndash2565 2001

[18] D P Dumas S R Caldwell J R Wild and F M RaushelldquoPurification and properties of the phosphotriesterase fromPseudomonas diminutardquo The Journal of Biological Chemistryvol 264 no 33 pp 19659ndash19665 1989

[19] A Mulchandani ldquoAmperometric thick-film strip electrodes formonitoring organophosphate nerve agents based on immobi-lized organophosphorus hydrolaserdquo Analytical Chemistry vol71 no 11 pp 2246ndash2249 1999

[20] N Jaffrezic-Renault ldquoNew trends in biosensors for organophos-phorus pesticidesrdquo Sensors vol 1 no 2 pp 60ndash74 2001

[21] B Prieto-Simon M Campas S Andreescu and J-L MartyldquoTrends in flow-based biosensing systems for pesticide assess-mentrdquo Sensors vol 6 no 10 pp 1161ndash1186 2006

[22] J Wang ldquoCarbon-nanotube based electrochemical biosensorsa reviewrdquo Electroanalysis vol 17 no 1 pp 7ndash14 2005

[23] R P Deo J Wang I Block et al ldquoDetermination oforganophosphate pesticides at a carbon nanotubeorganophos-phorus hydrolase electrochemical biosensorrdquo Analytica Chim-ica Acta vol 530 no 2 pp 185ndash189 2005

[24] D Herzlinger P Viitanen N Carrasco and H R KabackldquoMonoclonal antibodies against the lac carrier protein fromEscherichia coli 2 Binding studies with membrane vesicles andproteoliposomes reconstitutedwith purified lac carrier proteinrdquoBiochemistry vol 23 no 16 pp 3688ndash3693 1984

[25] T G Bernhardt and P A J de Boer ldquoThe Escherichia coli ami-dase AmiC is a periplasmic septal ring component exported viathe twin-arginine transport pathwayrdquo Molecular Microbiologyvol 48 no 5 pp 1171ndash1182 2003

[26] Y-H Liu Z-S Chen J Lian X Huang and Y-C ChungldquoPurification and characterization of a novel organophosphoruspesticide hydrolase from Penicillium lilacinum BP303rdquo Enzymeand Microbial Technology vol 34 no 3-4 pp 297ndash303 2004

[27] G R Chaudhry A N Ali and W B Wheeler ldquoIsolation ofa methyl parathion-degrading Pseudomonas sp that possessesDNA homologous to the opd gene from a Flavobacterium sprdquoApplied and EnvironmentalMicrobiology vol 54 no 2 pp 288ndash293 1988

[28] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[29] V Hooda A Gahlaut H Kumar and C S Pundir ldquoBiosensorbased on enzyme coupled PVC reaction cell for electrochemicalmeasurement of serum total cholesterolrdquo Sensors and ActuatorsB Chemical vol 136 no 1 pp 235ndash241 2009

[30] J H Lee J Y Park K Min H J Cha S S Choi and YJ Yoo ldquoA novel organophosphorus hydrolase-based biosensorusing mesoporous carbons and carbon black for the detectionof organophosphate nerve agentsrdquoBiosensors andBioelectronicsvol 25 no 7 pp 1566ndash1570 2010

[31] J Wang L Chen A Mulchandani P Mulchandani andW Chen ldquoRemote biosensor for in-situ monitoring oforganophosphate nerve agentsrdquo Electroanalysis vol 11 no 12pp 866ndash869 1999

[32] S H Chough A Mulchandani P Mulchandani W Chen JWang and K R Rogers ldquoOrganophosphorus hydrolase-basedamperometric sensor modulation of sensitivity and substrateselectivityrdquo Electroanalysis vol 14 no 4 pp 273ndash276 2002

[33] J Wang R Krause K Block M Musameh A Mulchandaniand M J Schoning ldquoFlow injection amperometric detectionof OP nerve agents based on an organophosphorus-hydrolasebiosensor detectorrdquo Biosensors and Bioelectronics vol 18 no 2-3 pp 255ndash260 2003

[34] V Sacks I Eshkenazi T Neufeld C Dosoretz and J RishponldquoImmobilized parathion hydrolase an amperometric sensor forparathionrdquo Analytical Chemistry vol 72 no 9 pp 2055ndash20582000

[35] T Laothanachareon V Champreda P Sritongkham M Soma-sundrum andW Surareungchai ldquoCross-linked enzyme crystalsof organophosphate hydrolase for electrochemical detection oforganophosphorus compoundsrdquoWorld Journal of Microbiologyand Biotechnology vol 24 no 12 pp 3049ndash3055 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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based on cholinesterase activity are also used for screeningprocesses But these methods are not well suited for processcontrol monitoring assays where fast and repetitive analysesare required [14]

These problems can be solved by the use of biosensortechnology which fulfils the demands for on-site monitoringand the rapid detection of neurotoxic agents [15] Biosensorsbased on the acetylcholine esterase (AChE) inhibition havebeen widely used for the determination of OP pesticides [16]The main drawback of inhibition-based biosensors involvesmultiple-step operation such as time-consuming incubationand reactivationregeneration steps [14 17]

Organophosphorus hydrolase (OPH) can catalyze thehydrolysis of a large number of OP pesticides [18] Theenzymatic hydrolysis of OP pesticides involves a pH changeas well as the generation of chromophoric species [19] OPH-based assays are direct and respond to OP pesticides assubstrate rather than inhibitors in case of inhibition-basedbiosensors Consequently these assays can be reversible andrequire only target analyte [14]Themain problem associatedwith these methods involves the use of free enzyme whichcan be used once and also increases the cost of procedureTo overcome this problem use of immobilizationmethod forconfining the enzyme molecules onto the different insolublesupports this enhances the reusability of enzyme throughimmobilization

Although OPH-based methods have several advantagessuch as selectivity for target analyte and show fast responsethey are less sensitive and have low detection limits ascompared to inhibition-based methods [20 21]

These problems can be overcome by using the carbonnanotubes (CNTs) as transducer material in amperometricbiosensors CNT showed excellent electrocatalytic propertieswith larger surface area [22] Deo et al which significantlyenhanced the amperometric signals generated from OPpesticides [23] The OPH biosensors provide a simple fastaccurate and selective monitoring of OP pesticides as wellas being good for in-field applications and monitoring ofdetoxification processes [19]

In the present method the OPH was extracted andpurified from Brevundimonas diminuta cells and used forthe development of electrochemical biosensor by covalentimmobilization of enzyme on to polyvinyl chloride (PVC)surface rather than working electrode This facilitates theopportunity to change the working electrode on deteriora-tion without wasting the immobilized enzyme which makesit economical and convenient The use of carbon nanotubesfor the construction of working electrode provides efficientcharge transfer from one phase to another The use ofpolyvinyl chloride (PVC) in bioanalytical techniques willopen a new pathway in biosensor design and fabricationdiagnostic kits and related biomedical instrumentation

2 Materials and Methods

21 Chemicals and Reagents Sephadex G-100 was purchasedfrom MP Biomedicals Nutrient broth tris-HCL EDTAlysozyme sucrose and sodium hydroxide were purchased

from Himedia Glutaraldehyde (grade 125) was purchasedfrom Sigma chemicals and organophosphorus (OP) pesti-cide (methyl parathion) was purchased from Shri Ram Fer-tilizers amp Chemicals All other chemicals were of analyticalreagent grade unless otherwise stated Double distilled water(DDW) was used throughout the experiments Silver wireand PVC (polyvinyl chloride) beaker were bought from localmarket

22 Instruments and Equipment Cyclic voltammetry (CV)measurements were performed on a potentiostatgalvanostat(PGSTAT1230302 Autolab) with a three-electrode systemcomposed of a platinum (Pt) wire as an auxiliary electrodean AgAgCl electrode as reference electrode and carbonnanotubes paste based working electrode UV spectropho-tometer (Shimadzu Corporation Japan) temperature con-trolled water bath (SunRon) digital pH meter (EUTECH)refrigerator (LG) microwave oven (LG) rotatory incubatorshaker (HICON) magnetic stirrer (HICON) refrigeratedcentrifuge (SIGMA) and deep freezer (Voltas) The study ofsurface morphology was carried out with scanning electronmicroscope (SEM) (Model Joel JSM-6510 Japan) at Depart-ment of Chemistry M D University Rohtak India

23 Microorganism The lyophilized sample of bacteria Bre-vundimonas diminuta (MTCC 3361) was purchased fromIMTECH Chandigarh

24 Revival and Growth of Bacteria Lyophilized sample ofB diminuta was mixed with 5mL of nutrient broth (pH-80)in a test tube to make a bacterial suspension The conicalflask (250mL) containing 100mL nutrient broth (pH-80)was inoculated with bacterial suspension The culture mediawere incubated in a rotatory incubator shaker at 200 rpm for24 hours at 30∘C Growth of bacteria was checked by takingOD at 600 nm

25 Extraction and Purification of OPH from Brevundimonasdiminuta After desirable growth the cells were centrifugedat 8000 rpm for 10 minutes The cell pellets was incubatedwith 075M Sucrose mixed in 75mL of 50mM SodiumPhosphate Buffer pH8 at 20∘C for 10minutesThe suspensionwar treated with lysozyme as described by Herzliger et al1984 [24] the suspension was centrifuged at 10000 rpm for 5minutesThe pallet was separated and re-suspended in 20mLof 50mM sodiumphosphate buffer (pH 8) containing 50mMNaCl at 4∘C for one hour with constant steering [25] Aftercentrifugation at 10000 rpm for 5 minutes the supernatantwas separated and treated as crud enzyme solutionThe crudesolution was concentrated using lyophilization machinePurification of concentrated crude Organophosphate hydro-lase was done using sephadex G-100 column (25 times 30 cm) asdescribed by Liu et al 2004 [26]

26 Enzyme Assay and Protein Estimation The 01mL ofpurified Organophosphate hydrolase enzyme solution wasmixed with 29mL Sodium Phosphate Buffer (pH 8) 5 nMmethyl parathion (01mL) was added to the enzyme solution





(120582 = 410 nm)(1)




4Cl (02mg)



AgAgCl electrode

Pt


PVC beaker































(a)

(a)

(b)

(b)


350

300

250

200

150

100

50

0

0 02 04 06 08 1 12 14

Curr

ent (120583

A)

Potential (V)


12

1

08

06

04

02

0

6 65 7 75 8 85 9 95 10

pH

Curr

ent (120583

A)



355 119870119898and 119868119898119886119909




(Figure 9)

12

1

08

06

04

02

0

Curr

ent (120583

A)

20 25 30 35 40 45 50 55 60

Temperature (∘C)


0 5 10 15 20


12

1

08

06

04

02

0

Curr

ent (120583

A)


12

1

08

06

04

02

0

Curr

ent (120583

A)

0 50 100 150 200 250 300 350 400 450 500




25

20

15

10

5

0

minus5minus002 0 002 004 006 008 01 012

1[I]

1[S]






5 493 plusmn 002 98610 991 plusmn 002 991












30

25

20

15

10

5

00 5 10 15 20 25 30M

ethy

l par

athi

on co

ncen

trat

ion

(120583M

)in

spik

ed w

ater

by

pres

ent m

etho

d


R2= 0985


12

1

08

06

04

02

0

Curr

ent (120583

A)

1 5 10 15 20 25

Number of uses





12

1

08

06

04

02

0

Curr

ent (120583

A)

0 10 20 30 40 50










4 Conclusion





Acknowledgment


References















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of





(120582 = 410 nm)(1)




4Cl (02mg)



AgAgCl electrode

Pt


PVC beaker































(a)

(a)

(b)

(b)


350

300

250

200

150

100

50

0

0 02 04 06 08 1 12 14

Curr

ent (120583

A)

Potential (V)


12

1

08

06

04

02

0

6 65 7 75 8 85 9 95 10

pH

Curr

ent (120583

A)



355 119870119898and 119868119898119886119909




(Figure 9)

12

1

08

06

04

02

0

Curr

ent (120583

A)

20 25 30 35 40 45 50 55 60

Temperature (∘C)


0 5 10 15 20


12

1

08

06

04

02

0

Curr

ent (120583

A)


12

1

08

06

04

02

0

Curr

ent (120583

A)

0 50 100 150 200 250 300 350 400 450 500




25

20

15

10

5

0

minus5minus002 0 002 004 006 008 01 012

1[I]

1[S]






5 493 plusmn 002 98610 991 plusmn 002 991












30

25

20

15

10

5

00 5 10 15 20 25 30M

ethy

l par

athi

on co

ncen

trat

ion

(120583M

)in

spik

ed w

ater

by

pres

ent m

etho

d


R2= 0985


12

1

08

06

04

02

0

Curr

ent (120583

A)

1 5 10 15 20 25

Number of uses





12

1

08

06

04

02

0

Curr

ent (120583

A)

0 10 20 30 40 50










4 Conclusion





Acknowledgment


References















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



















(a)

(a)

(b)

(b)


350

300

250

200

150

100

50

0

0 02 04 06 08 1 12 14

Curr

ent (120583

A)

Potential (V)


12

1

08

06

04

02

0

6 65 7 75 8 85 9 95 10

pH

Curr

ent (120583

A)



355 119870119898and 119868119898119886119909




(Figure 9)

12

1

08

06

04

02

0

Curr

ent (120583

A)

20 25 30 35 40 45 50 55 60

Temperature (∘C)


0 5 10 15 20


12

1

08

06

04

02

0

Curr

ent (120583

A)


12

1

08

06

04

02

0

Curr

ent (120583

A)

0 50 100 150 200 250 300 350 400 450 500




25

20

15

10

5

0

minus5minus002 0 002 004 006 008 01 012

1[I]

1[S]






5 493 plusmn 002 98610 991 plusmn 002 991












30

25

20

15

10

5

00 5 10 15 20 25 30M

ethy

l par

athi

on co

ncen

trat

ion

(120583M

)in

spik

ed w

ater

by

pres

ent m

etho

d


R2= 0985


12

1

08

06

04

02

0

Curr

ent (120583

A)

1 5 10 15 20 25

Number of uses





12

1

08

06

04

02

0

Curr

ent (120583

A)

0 10 20 30 40 50










4 Conclusion





Acknowledgment


References















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


25

20

15

10

5

0

minus5minus002 0 002 004 006 008 01 012

1[I]

1[S]






5 493 plusmn 002 98610 991 plusmn 002 991












30

25

20

15

10

5

00 5 10 15 20 25 30M

ethy

l par

athi

on co

ncen

trat

ion

(120583M

)in

spik

ed w

ater

by

pres

ent m

etho

d


R2= 0985


12

1

08

06

04

02

0

Curr

ent (120583

A)

1 5 10 15 20 25

Number of uses





12

1

08

06

04

02

0

Curr

ent (120583

A)

0 10 20 30 40 50










4 Conclusion





Acknowledgment


References















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


12

1

08

06

04

02

0

Curr

ent (120583

A)

0 10 20 30 40 50










4 Conclusion





Acknowledgment


References















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of




































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

research article preparation of electrochemical biosensor...

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