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ImmobilizationoftheureaseoneggshellmembraneanditsapplicationinbiosensorS.F.D'Souza⁎,JitendraKumar,SandeepKumarJha,B.S.KubalNuclearAgricultureandBiotechnologyDivision,BhabhaAtomicResearchCentreTrombay,Mumbai-400085,IndiaabstractarticleinfoArticlehistory:Received8June2012Receivedinrevisedform24October2012Accepted8November2012Availableonline17November2012Keywords:PolyethyleneimineEggshellmembraneUreasePotentiometricureabiosensorEggshellmembraneisanaturalmaterial,essentiallymadeupofproteinfibershavingflexibilityintheaqueoussolutionandpossessinggasandwaterpermeability.Itisusedasabiomembraneforimmobilizationofureaseforthedevelopmentofapotentiometricureabiosensor.Eggshellmembranewastreatedwithpolyethyleneimine(PEI)toimpartpolycationcharacteristics.UreasewasimmobilizedonthePEItreatedegg-shellmembranethroughadsorption.SEMstudywascarriedouttoobservethechangesinsurfacemorphologyafterimmobilization.FTIRstudyofmembranewascarriedouttoobservethechangesinIRspectraafterimmobilizationofenzyme.Immobilizedmembranewasassociatedwithammoniumionselectiveelectrode.Biosensorexhibitedsigmoidalresponsesfortheureaconcentrationrangefrom0.5to10mM.Theresponsetimeofthebiosensorwas120s.Asinglemembranewasreusedfor270reactionswithoutlossofactivity.Theurease–eggshellmembraneswerestablefor2monthswhenstoredinbufferevenatroomtemperature.©2012ElsevierB.V.Allrightsreserved.1.IntroductionAlargenumberoftechniquesandmaterialshavebeenusedfortheimmobilizationofbiocatalystforbiosensorapplication[1–12].Thechoiceofmaterialsandthetechnique,forthepreparationofmembranes,hasbeendictatedbythelowdiffusionresistanceofthemembranecoupledwithitsabilitytoincorporateoptimalamountofenzymeperunitarea.Thetechniques,usedforimmobilization,shouldleadtominimalinactivationoftheenzyme.Also,forasuccessfulbiosensor,theimmobilizedenzymemembraneshouldbestableformultipleuseaswellasstorage.Avarietyofsyntheticaswellasnaturalpolymericmaterialshavebeenusedfortheimmobilizationofenzymesforbiosensorpreparation.Amongthesenaturalmaterials,especiallyofproteinicorigin,haveshownpromiseuses.Oneofthesenaturalmaterials,onionmembrane,hasalreadyprovenitsapplicationforbiosensor[7,9].Othersuchmembranewhichhascurrentlygainedimportanceistheeggshellmembrane.Eggshellmembraneisalsoanaturalmaterial,essentiallymadeupofproteinfibers,havingflexibilityintheaqueoussolutionandpossessingexcellentgasandwaterperme-ability.Itisalightpinkdouble-layeredmembraneinsidetheeggshellcomposedofhighlycross-linkedproteinssimilartokeratin,collagenandelastin[13].SomereportsonbiosensorshavebeenpublishedusingeggshellmembraneasasupportingmatrixforimmobilizationofenzymessuchasD-aminooxidase,catalase,myrosinase,tyrosinaseandglucoseoxidase[12,14–19].Thetechnique,whichmostlyusedforimmobilizationofenzymeoneggshellmembrane,involvedadsorptionfollowedbycross-linkingwithglutaraldehyde.Glutaraldehydehasbeenusedforfixingtheenzymetothesupport.However,glutaraldehydecross-linkingoftenreducestheenzymeactivityafterimmobilization[20].Thiscanbeobviatedbyimmobilizationoftheenzymethroughadsorptiononchargedpolymericsupports.Extensivestudieshavebeencarriedoutinourlaboratoryforimpartingpolycationiccharacter-isticstoavarietyofpolymericmaterialsusingpolyethyleneimine(PEI)fortheimmobilizationofenzymesorcells[21–28].Ureaisaby-product,whichisoftenmonitoredinbloodtoobtaininformationonkidneydisease.Itisgenerallyacceptedtobethebestmarkerforevaluatingthelevelofuremictoxins[29].Urease(E.C.3.5.1.5)promotesthereactioninvolvingthedecom-positionofureatogiveammoniaandcarbondioxide.NH2CONH2þ3H2O→urease2NHþ4þHCO3þOHGuilbaultandMontalvowerethefirsttofabricateapotentiometricureaseenzymeelectrodefortheestimationofureathroughitsenzyme-catalyzedhydrolysis[30,31].Inonereview,Dhawanetal.[32],reportedtherecentdevelopmentsofureabiosensor.Manypotentiometricureabiosensorsbasedonthedetectionofammoniumionproducedbytheaboveenzymaticreactionhavebeenreported[33–36].Glutaraldehydecross-linkedureasehasshownonly15timesoperationalreusabilityforbiosensorapplication[37].Inonereport,apotentiometricureabiosensorwasdevelopedbasedonmodifiedelectrodeswithureaseimmobilizedonPEIfilmsthatexhibitedsigmoidalresponsesfortheureaconcentrationworkingrangefrom1×10−2.5to1×10−1.5Mandalifetimeof4weeks[36].Theaimofthepresentworkwastoprepareastableurease–eggshellmembranethroughadsorptionusingPEIforbiosensorapplication.MaterialsScienceandEngineeringC33(2013)850–854⁎Correspondingauthor.Tel.:[phone];fax:[phone].E-mailaddresses:[e-mail](S.F.D'Souza),[e-mail](J.Kumar).0928-4931/$–seefrontmatter©2012ElsevierB.V.Allrightsreserved.:(PEI)andjackbeanurease(E.C.3.5.1.5)(910μMunitspertablets)wereobtainedfromSigmaChem.Co.USA.Allotherchemicalsusedwereofanalyticalreagentgrade,obtainedfromstan-dardso