纳米颗粒模拟酶及应用(3)

研究了纳米颗粒的多酶活性,包括过氧化物酶和过氧化氢酶,以及在葡萄糖高灵敏检测中的应用.

Zhangetal.PrussianBlueModi edFerritinasPeroxidaseMimeticsandItsApplicationsinBiologicalDetection

meaningoftheMichaelisconstantKrateofconversionmisthesubstratecon-centrationatwhichtheishalfofVmax,enzymeswithsmallerKmhavehigheraf nitywiththesubstrates.

2.3.3.PB-FtDependenceNPsConcentration,ofPeroxidase-LikepHandActivityTemperature

ThecatalyticreactionwascarriedunderdifferentpH,tem-peraturesandPB-FtNPsconcentration.PB-FtNPscon-centrationwasfrom1.012 g/mLto7.092 g/ml,thepHchangedfrom2to11,thetemperatureincreasedgradu-allyfrom10 Cto75 C.Accordingtothecolorimet-ricreactionofTMBandABTSoxidizedbyH2O2,thesedependenceswererecordedbytheabsorbancevalueofthereactionsystemsusingShimadzuUV-3600UV-VIS-NIRspectrophotometer.2.4.GlucoseDetection

Glucosedetectionwasperformedasfollows:(a)100 Lofglucoseofdifferentconcentrationswasincubatedat37 Cfor45minwith100 L0.58mg/mLGOxin100 LPBS(pH=7 4);(b)100 LABTSand150 LPB-FtNPsin450 LNa2HPO4–C6H8O7buffer(pH=3 0)werethenaddedin,thesolutionwasincubatedat40 Cfor45min;(c)Finally,theabsorbanceat415nmofthemixedsolutionwasmeasuredbytheUV-VIS-NIRspectrophotometer.2.5.Enzyme-LinkedImmunosorbentAssay(ELISA)Thespeci ctargetingabilityofPB-FtNPswasveri edbytheELISA.Theexperimentwascarriedoutin48-wellplates,100 Lof10 g/mLanti-HoSFinPBSbuffer(pH=7 4)wascoatedtothewellsurfaceat37 Cfor1handthenkeptat4 Cfor12h,next,thewellswerewashedthreetimeswithPBS-T(pH=7 4,Tween-200.05vol-ume%),eachfor5min.Afterthat,theplatewasblockedwith150 L1%BSAperwellat37 Cfor2htopreventthenon-speci cbindingofimmunoglobulins,thewellswerethenwashedagainwithPBS-Tasdescribedbeforeandaddedwith100 LPB-FtNPs.(Thecontrol-1andthecontrol-2groupwereobtainedbyidenticaltreatmentwith100 LPBSandHoSF,insteadofPB-FtNPs),afterincubatedat37 Cfor2handwashedbyPBS-Tforthreetimes,thewellswere lledwithchromogenicsubstrateTMBandH2O2,andtheabsorbanceat650nmwasmea-suredbyamicroplatereader(Elx808,USA).Inthestudy,concentrationdependencyofperoxidase-likeactivitywasmeasuredbydilutingPB-FtNPsinHCl(pH=3 0)atdou-blingdilutions(1:2,1:4,1:8,1:16,1:32,1:64),highandlowanti-HoSFlevels(1 g/mL,2.5 g/mL,5 g/mL,10 g/mL)usingtocoatthewellwerealsodiscussedinthisstudy.

J.Nanosci.Nanotechnol.12,1–8,2012

3.RESULTSANDDISCUSSION

3.1.CharacterizationofPB-FtNPs

TheobtainedPB-FtNPsdisplaysimilarinnercoremor-phologytothepureferritin,asshowninTEMimages(Figs.2(a),(c)).FromtheTEMimagenegatively-stainedwith2%phosphotungsticacid,itcanbeobservedthatthesizeandmorphologyoftheferritinshellremainedthesamewithferritinsbeforereaction(Fig.2(b),(d)),whichindicatedthatthesynthesisprocessofthePBNPsdidnotchangethesizeandmorphologyofthehollowsphericalshellofferritin.Fromthehigh-resolutionTEM(HRTEM)images(insetsofFig.2(a),(b))wefoundthatthecrystalstructureoftheironoxidecoreswasdisruptedtosomeextentafterthereaction,however,theimagesshowtheregularlatticefringesremained0.25nm,whichisrela-tivetothe(110)planesofferrihydrite.35UV-visabsorptionspectraofPB-FtNPsinwater(Fig.3)presentedPB’schar-acteristicpeakatapproximately700nm36andtheabsorp-tionofferritinat280nm,resultingfromtheformationofPBNPsinthepresenceofferritin.Notethat,noindividualPrussianblueparticleswereobservedintheTEMsam-ple,asshowntypicallyinFigure2(c).Therefore,itcouldbededucedthatPrussianbluewasdepositedonthesur-faceoftheironoxidecoresratherthanformedinreactionsolution,butitwashardlydistinguishedfromironoxideduetolowcrystallinityandverysmallsize.DynamiclightscatteringanalysisdemonstratedthattheHoSFdissolvedinaqueoussolutionofpH=3 0haveameansizeof10.9nm(Fig.2(e)),whichisclosetothediameterofHoSF,indicatingferritinismonodispersed.Aftermodi -cationwithPB,thesizesofthenanoparticlesincreasedto22.8nm(Fig.2(f)),attributedlikelytotheaggregationoftheformedPB-FtNPsinducedbysurfacePBdepositionandchargevariation.ZetapotentialofHoSFis+23 8mVinpH3.0,accordingwiththefactthatHoSFhasaniso-electricpoint(IEP)atpH5.8–6.37WhereaszetapotentialofPB-FtNPsis+13 7mV,indicatingthatthenegativechargeofPBcoatedontheironoxidecoresreducedthepositivechargeofHoSF.

3.2.ThePeroxidase-LikeActivityofPB-FtNPsTheperoxidase-likeactivityofPB-FtNPswasevalu-atedthroughacolormetricreactionusingTMBorABTSassubstratesinthepresenceofH2O2.AsshowninFigure4(A),theobviouscolorchangescanbeseenwhenPB-FtNPswereusedascatalyst.TheabsorbancechangeofoxidizedproductsofsubstrateswasalsomonitoredwithreactiontimeusingHoSFandPB-FtNPsascatalysts(Figs.4(B),(C)).HoSFdisplaysalmostnoperoxidase-likeactivitywhenthechromogenicsubstrateisTMBbutweakactivitywhenusingABTSaschromogenicsub-strate.ThereasonmaybethatferritinsareelectropositiveandABTSisnegativelychargedwhileTMBispositivelychargedinNaAc-HAcbuffer(pH=3 6),resultinginthe

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