The Photodestruction of Virus in Nano-TiO2 Suspension

1、The Photodestruction of Virus in Nano-TiO2 Suspension422DOIl0.1007/sll595.006.3422.6Vo1.22No.3XURuifenetal:ThePhotodestructionofHrusinNano-TiOeSuspensionThePhotodestruetionofVirusinNano-TiO2SuspensionxuRuifen,LIUXil11)l!,zHANGP蛐g2,MAHaoLIUG蛐g2,XIAZhengyan(1?CollegeofMaterialsScienceandEngineering,Be

2、ijingUniversityofChemicalTechnology,Beijing100029.China;2.CollegeofLifeScienceandTechnology,BeijingUniversityofChemicalTechnology,Beijing100029,China;3.NingboGreen.EnjoyNanoTechnologyR&DCOLTD.,Ningbo315300,China)Abstract:HepatitisBsurfaceantigen(HBsAg)wasselectedasreferencetoevaluatethephotodest

3、ructiveeffectofaself-preparednanoTiO,onvirusesinaqueoussuspensionthroughsandwichELISAassay(aninvitroenzymeimmunoassay)underdifferentconditions,andmoregeneralexperimentsonRNA(ribonucleicacid)andcaseinwerecarriedout.ResultsindicatethatTiO.isdestructiveatleasttomostvirusesinwater.Themechanismofdestruct

4、ionwasdiscussed.Keywords:nanoTiO,;photodestruction;HbsAg;virus1IntroductionTiO,isasafe,stableandemcientsemiconductorphotocatalystunderintensiveresearchforitscapabilityofsolarenergyconversionandthepotentialapplicationsinsolarcell,watersplitting,environmentalcleanupandetc【.Itsphotodestructionofmicroor

5、ganismsisabigbranch.Currently,therearealreadyalotofreportsonthebactericidaleffectofTiO,inaqueoussuspensionssuchasonEscherichiacoli,LactobacillusacidophilusandStaphylococcusaureusJ.anditwasfoundthatthecomeintocontactwithTiO,andthusdeservespecificinvestigation.SinceHepatitisBvirus(HBV)isarepresentativ

6、einfectiousvirusharassingaboutl/l0ofthepopulationinChina,inthisstudy,weselectedHBsAg,themaincomponentOfHBVcapsid,asthereferencetoiudgethephotodestructionofvirusinnano-TiO,suspension.andfurtheronamoregeneralbackground.thephotodestructionOfRNAandcaseinwasalsoinvestigated.2Experimentalphotokillingactiv

7、itywasassociatedwiththereduction2.1MaterialsinthelevelofintracellularcoenzymeA(CoA)throughphotooxidationthusthecellrespirationwasinhibitedNano-TiO2powder(self-prepared:thesurfacewasmodifiedwithaninorganiccoating;themeanandthemicrobeswereledtodeath.However,toourdiameterwas24nm;whendispersedinaqueousm

8、edia,knowledge,thereportsonthephotokillingofvirusesaremuchmorescarcelyseen,exceptforseveralPapersonphageMS2rasingle-strandedRNAbacteriophage)andpoliovirusl【.Comparedwithbacteria.virusesaremuchsmallerinsizer10-300nm)andcanpassthroughfiltersthatretardmostofthebacteria,besides,viruseshavenocellularstru

9、cturebutaremainlycomposedofanucleiccoreandaproteincapsid;itcanbeexpectedthatvirusesresponddifferentlyfrombacteriawhen(Received:March13,2006;Accepted:Apr.14,2007)XURuifen(徐瑞芬1:Assoc.Pro;Email:.CnFundedbyNationalKeyScienceandTechnologyAchievementsPromotionProgram(No.2003EC000039)ofthe

10、MinistryofScienceandTechnology,China88%ofthesoftaggregatesweresmallerthan100nmandthemostprobablesizewas66nm);purifiedHBsAgsolution(4mg/mL,suppliedbyBeijingMDCBiotechnologyCo.,Ltd.);ultrapurewater;RNA(BR);casein(BR).Bio-RAD550microplateELISAreader(BioRAD,Hercules,CA);UV-2000ultravioletspecteophotomet

11、er(UNICAN,Shanghai);TDL-5-Ahigh-speedcentrifuge(ShanghaiAntingInstrumentFactory);160Wself-ballastingmercuryvaporlamp(BeijingHuatePhotoelectricityEquipmentFactory).2.2AssayoftheHBsAgphotodestructionThepurifiedHBsAgsolutionwasdilutedwithultrapurewaterto0.1mg/mL;thisconcentrationis10000timesthesensitiv

12、ityoftheELISAkitandJournalofWuhanUniversityofTechnologyMater.Sci.Ed.Sept2007satisfiesthemeasurementrequirement.ThenacertainamountoftheTiO,powderwasintroduced.underasepticcondition,into5mLofthedilutedHBsAgsolution,andwasshakenforacertainperiodat2O.Thesuspensionwasthencentrifugallysettledr5000rpm,5min

13、),thesupernatantwascollectedandtheOD(opticaldensity)valuewasmeasuredbysandwichELISAassayonELISAreader.Theexperimentswereconductedintriplicate.ControlexperimentswereruninparallelwithoutTi0,.FromthemeanODofthethreemeasurements,S/Nratio(OD(sample)/OD(negativecontrol,inthisexperiment,was0.O21)wascalcula

14、ted.Iftheratioisequaltoorhigherthan2.1.theresultisconsideredtobeHBsAgpositive(+),andinreverse,isconsideredtobeHBsAgnegative().Whentheblankcontrolsamplegivespositiveresultwhilethetestsamplegivesnegativeresult,wesaythatthepowderinthesuspensionisdestructivetotheHBsAg.3ResuitsandDiscussion3.1Theestablis

15、hmentoftheassaymethodVirusesaremainlycomposedofanucleicacid(DNAorRNA)coreandaproteincapsid.Thecapsidconstitutesaprotectivecoveringaroundtheviralgenomeandinthecapsidthereisanattachmentproteinnecessaryforthevirustoattachtoitstargetcellbeforeitcanenterintothatcel1.Obviously,ifthecapsidisdestroyed,onone

16、hand,thecorewillbemucheasiertobeattacked.andontheother,theviruswillhavenowaytouptakeintosusceptiblecellsforthelossofthespikes.HBVisawidespreadvirusandHBsAg(hepatitisBsurfaceantigen)isthemaincomponentinitscapsid.thedestructionOfHBsAgcreateschancesforTiO,toactontheDNAcoreandalsohelpstopreventHBVfromin

17、vadingintohostcells.Forthisreason,inthiswork,weselectedHBsAgtoevaluatethephotodestructione毹ctOfTiO,onviruses.ThedestructionexperimentsonHBsAgaregenerallyconductedinphosphatebuffer(PBS)solutioncontaining1O%ofbovinecalfserum(BCS).howeveLsinceTiO,candegradeBCStosomeextent,theresultwillbeunauthentic.Thr

18、oughcomparison,wefinallychoseultrapurewaterassolventtodissolveHBsAg.ThepurposeofthecentrifugalprocessistoremoveTiO,soastominimizemeasurementinterferenceanderror.NocentrifugaldestructionOfHBsAgwasobservedwhencentrifugedandnoncentrifugedsampleswerecompared.4233.2TIhephotodestruetionlevelsofHBsAgunderd

19、ifferenteonditionsThedestructionlevelsofHBsAgatdifferentTi0,concentration,differentcontacttimeanddifferentlightsourcewereinvestigated.3.2.1TheinfluenceofTiOconcentrationTheexperimentsontheeffectofTiOconcentrationonthephotodestructionofHBsAgwerecarriedoutundernaturallighting(insidewatchglass,whereUVl

20、ightsarealmostunavailable)for12handtheresultsaregiveninTable1.Fromthetable.itcanbeseenthatwiththeincreaseoftheTiO,concentration.thedestructionlevelofHBsAgisincreased.Whentheconcentration01Osverysmal1.theSNratiodirslittlefromthatoftheblankcontro1.andwhentheconcentrationincreasesto1%.theresultischange

21、dfrompositivetonegative.3.2.2TheinfluenceofcontacttimeFable1ThecorrelationbetweenTiO2concentrationandHBsAgph0t0destructi0nTiO2concenlrafion/o00.02.0S/N142.9(+)123.8(+)88.2(+)4.58(+)2.05(-)1.57(-)Note一indicatespositiveand一indicatesnegative;theODofthestandardnegativecontrolis0021:similarlyhe

22、reinailerTheexperimentsonthee毹ctOfcontacttimeonthephotodestructionofHBsAgwerecarriedoutundernaturallightingasmentionedaboveand1%OfTiO,concentration.AsshowninTlab1e2.withtheextensionofthetime.theHBsAgisgraduallydestroyed.Atier12hcontact,theHBsAgresultischangedfrompositivetonegative.indicatingthattheT

23、iO,canwelldestroyHBsAginarathershorttimeperiodandtheminimumlethaltimeis12hwhentheconcentrationis1%.Table2rhecorrelationbetweenconctacttimeandHBsAgph0t0destructi0n3.2.3TheinfluenceoflightsourceThephOtOdestructiOnlevelsofHBsAgunderdifferentlightsourcesincludingUVirradiation.16OWhighpressuremercurylamp

24、irradiation.naturallightingandweaklighting(inthedarkwhilenotopticallyfiltered)wereinvestigated;theTiO,concentrationusedherewas1%andthecontacttimewas12h.ThecorrespondingresultsaregiveninTable3.Table3ThecorrelationbetweenexcitationlightsourceandIIBsAgph0t0destructi0nItisshownthattheTiO2,ataproperconce

25、ntration424Vo1.22No.3XURuifenetal:ThePhotodestructionofVirusinNanoTiO2Suspensionandcontacttime,isdestructivetoHBsAgunderanyofUVirradiation,mercurylamplighting,naturallightingandweaklighting,althoughinadegressiveorder,indicatingthattheTiO2isphOtOcatalyticallyactiveunderalltheselightsourceconditions.3

26、.3ThephotocatalyticoxidationofHBsAgChlorineandozonearetwoeffectiveoxidantsusuallyusedtoinactivateviruses.Accordingtoliterature5,chlorineinactivatesvirusesbyoxidationofthecapsidornucleicacids,andozoneoxidizestheviralcapsid,whichsubsequentlyinterfereswiththeabilityofvirusestoinvadehostcells.Comparedwi

27、ththem,whendispersedinaqueousmedia,TiO2particlescanessentiallybethoughtofasshortcircuitedphotoelectrochemicalcellsandredoxreactionscanoccuronthesephotoanodesandphotocathodesiftheirradiationwithanappropriateenergyisgivenandelectronholepairs(e/h)areexcited【.Sincetheredoxpotentialatjtsvalancebandis+3.0

28、V(vsNHE,pH=7),TiO2isthermodynamicallycapableofdestroyingmanysubstancesincludingHBsAgdirectlythroughholeinjection,functioningsimilarlyaschlorineandozone.Additionally,inaqueousmedia,thephotoexcitedholesandelectronsmayfurtherundergothefollowingreactionswithhydroxideions,wateranddissolvedoxygen:h+OH.?OH

29、h+H2O?OH+He.+O2?O厂?(one.electronreductionofO2)2e.+O2+2HH2O2(twoelectronreductionofO2)3e.+O2+3HH20+?OH(three.electronreductionofO,)bystrongoxidants.ThepositivenegativechangeinHBsAgactivityintheaboveexperimentsmaybetheresultofboththephotoexcitedholesandthehydroxylradicalsgeneratedfromholesandelectrons

30、.Thephotodestructionofcaseinasdescribedinsection3.4isanotherexampleofproteindestruction.ThenaturallightingandweaklightingresponsivenatureoftheproductmaycomefromthesurfacemodificationofpurenanoTiO2withinorganiccoating;thiswillbediscussedindetailinanotherPaDer.&4Thephotodestruetion0fRNAandcaseinSi

31、ncevirusesarecommoninthattheyareallcomposedmainlyofproteinsandanucleicacid(DNAorRNA),onamoregeneralbackground,wealsotestedthephOtOdestructiOneffectsofnano.TiO,onRNAandcasein.ThedestructionofRNAwasevaluatedbyintroducingacertainamountOfTiO,intoRNAsolution,centrifugallyremovingTiO2afterthereaction,dete

32、rminingtheRNAconcentrationbeforeandafterthereactionthroughUVabsorptionmeasurementandthusgainingthedestructionrate.Thedestructionexperimentoncaseinwassimilarexceptthatthedeterminationofcaseinconcentrationbeforeandafterthereactionwasdonebybiuretmethod.Theexperimentalconditionsforthebothwereasfollows:l

33、%ofTi0,concentration.highpressuremercurylamplighting,and24hcontact.Table4Theph0t0destructi0nofRNAandcaseinRNACaseinDestructionRate/%706()TheresultsaregiveninTable4.Weknowthatpyrimidineandpurineareimportantmolecularstructureunitsofnucleicacid.Theyarenitrogencontainingheterocycliccompoundswithconjugat

34、eddoublebonds.Thehydroxylradicals?O厂?+H2O2OH.+O2+?OH(Harber-Weissmayeasilyundergoanadditionreactionwith-Nreaction)Inthisway,hydroxylradicals(?OH)arecreated.Theunpairedelectroninthe?OHhasaverystrongtendencytobecomepaired,thusitexhibitsaverystrongreactivity,theredoxpotentialis+2.8V(NHE,pH=7),andcanalm

35、ostcompletelymineralizeallkindsoforganicstoCO2,H2O,SO4,C1一andetc.TheexistenceofhydroxylradicalsinTiO2aqueoussuspensionhasbeenprovedbymanyresearchesthroughelectronspinresonance(ESR)spintraptechnique引.fluorescencemethod.colorimetricmethodIoJandetc.HBsAg,asaprotein,isassembledfromaminoacidsthroughpepti

36、debonds.The_NH3,一CONH一,一OH.一SHandthelikeinitarealleasilyattackablegroups,producingpyrimidineandpurineradicals.Inthepresenceofoxygen,theseradicalsmayundergofurtherradicalreactions,thusdamagethepyrimidine/Durineandevencausethebreakageofthestrands.Furthermore,thehydroxylradicalscanabstracthydrogenfromr

37、ibose(ordeoxyribose)andchangethestructureofthelatter【,thuspreventingpyrimidine/purineandribose(ordeoxyribose)fromformingglycosidicbond.Inthisway,thelivingsubstancesofvirusesaredamagedandthusthevirusesarejnactivated.4ConclusionsWhendispersedinaqueousmedia,withenoughJournalofWuhanUniversityofTechnolog

38、yMater.Sci.Ed.Sept2007c0ncentrati0nandcontacttime.thetestednanoTiO,iSdestructivetoHBsAg.RNAandcasein.TiO,iSdestructiveatleasttomostvirusesinwater.SinceTiOhasthecombinedfunctionsofpollutantdegradationandmicrobialsterilization.theadvantageofalmostcompletelymineralizingorganicsandiSsafeandnontoxictohumanbody.itcanbeusedinwatertreatmentmedicalappliancesdisinfectionandsomeotherfieldsformultipurposeatthesametime.Referencesf11AMills,SLHunte.AnOveryiewofSemiconductorPhotocata1ysis.t,.Photochem.Photobio1.,A,1997,108:135f21TMatsunaga,RTomoda,TNakajima,eta1.Continuoussterilization