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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,especially