纳米颗粒模拟酶及应用(2)

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.

PrussianBlueModi edFerritinasPeroxidaseMimeticsandItsApplicationsinBiologicalDetectionZhangetal.

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LCITRAHCRAESERcoresthusprovidingrelativelyhighenzymemimeticactiv-ity;meanwhile,weintendedtopreservethefunctionofferritininthePrussianbluemodi edferritinnanoparticles(PB-FtNPs).Thenanoparticlesenzymemimeticactivity tswelltheMichaelis–Mentenequation,whichmakePB-FtNPsagoodreagentinglucosedetection.Theconse-quenceofenzyme-linkedimmunosorbentassay(ELISA)revealsthespeci cityofferritinwaspreservedinPB-FtNPsanditmakesPB-FtNPsausefulmimicenzymelabelinbiomedicaldetection.

2.MATERIALSANDMETHODS

2.1.Materials

Allchemicalsusedinthisexperimentwereofanalyti-calreagentgrade.Deionizedwaterwasusedthroughoutthestudy.Horsespleenferritin(HoSF),anti-horsespleenferritinantibody(anti-HoSF),3,3 ,5,5 -tetramethylbenzi-dine(TMB)and2,2 -Azinobis-(3-ethylbenzthiazoline-6-sulphonate)(ABTS)wereboughtfromSigmaAldrichCo.Ltd.(USA).Dimethylsulfoxide(DMSO),glacialaceticacid,anhydroussodiumacetate,citricacidmono-hydrate,sodiumsul te,hydrochloricacid,hydroxylam-moniumchloride,1,10-phenanthroline,dimethylbenzene,nutralbalsamandethylalcoholwerepurchasedfromSinopharmChemicalReagentCo.,Ltd.(China).Hydro-genperoxide(30%),glucoseandglucoseoxidase(GOx)wereobtainedfromAladdinCo.,Ltd.(China).SodiumphosphatedibasicandpotassiumhexacyanoferratewerereagentsfromShanghaiLingfengChemicalReagentCo.,Ltd.(China).SodiumbicarbonatewaspurchasedfromShanghaiHongguangChemicalFactoryCo.,Ltd.(China).SodiumhydroxidewasobtainedfromGuangzhouGuanghuaChemicalFactoryCo.,Ltd.(China).BovineSerumAlbumin(BSA)wasobtainedfromNanjingBook-manBiotechnologyLtd.(China).

2.2.SynthesisandCharacterizationofPB-FtNPsInourexperiment,anoptimalmolarratioofK4Fe(CN)HoSF=0 75 1wasusedtosynthesizePB-FtNPs.20 6/Lof47mg/mLHoSFwasmixedwith20mLHClsolution(pH=3 0),theconcentrationofiron(III)containedintheironoxidecoresisapproximately0.025Minthismixture;150 L0.025MK4Fe(CN)6wasaddedtotheformersolu-tionunderstirring,thenthesolutionwasmovedto4 Cfor12h.Afterthat,PB-FtNPswereobtainedafterdialysisandkeptunder4 Cforlaterexperiments.ThesynthesisprocedureisasillustratedinFigure1.

ThemorphologyandparticlesizeofPB-FtNPswerecharacterizedbytransmissionelectronmicroscopy(TEM,JEOLJEM-2100)at120kV.Dynamiclightscattering(DLS)togetherwithaParticleSizingAna-lyzer(Brookhaven,ZetaPlus)wereusedtodeter-minehydrodynamicsizedistributionofthenanoparticles.2

Fig.1.SchematicillustrationofthesynthesisrouteofPB-FtNPs.ThereactionofK4Fe(CN)6withiron(III)ontheironoxidecoregiverisetotheformationofPBNPswithintheferritincavity.

ShimadzuUV-3600UV-VIS-NIRspectrophotometerwasusedtomeasuretheUltraviolet-visible(UV-vis)absorptionspectraofthenanoparticles.

2.3.Peroxidase-LikeActivityofPB-FtNPs2.3.1.TestofPeroxidase-LikeActivityofPB-FtNPsThecatalyticexperimentofPB-FtNPswascarriedoutatroomtemperatureinareactionsystemwith400 L0.05mg/mLPB-FtNPsin4mLNaAc-HAcbuffer(pH=3 6)inthepresenceof320 L10mg/mLTMB(orABTS)and640 L30%H2O2assubstrates.TheabsorbancevaluesofthereactionsystemscatalyzedbyPB-FtNPswererecordedfor10minwiththemicroplatereader.

2.3.2.TheMichaelis–MentenKineticAnalysis

Steady-statekineticassayswerecarriedoutat25 Cwith10 L0.05mg/mL(theferritinconcentration)PB-FtNPsin200 LNaAc-HAcbuffer(0.2M,pH=3 6)inthepresenceofH2O2andTMB(orABTS)in96-wellplates.ThekineticanalysisofTMB(orABTS)asthesubstratewasperformedbyadding32 L30%Hamounts(0,0.5,1,2,4,6,8,10 L)2Oof2anddiffer-entTMBsolu-tion(10mg/mL,dissolvedinDMSO)orABTSsolution(10mg/mL,dissolvedindeionizedwater).ThekineticassayofH2O2asthesubstratewasperformedbyadding10 LTMBanddifferentamounts(2,4,8,12,16,32,64 L)of1%H2O2solution,or10 LABTSanddif-ferentamounts(2,4,8,12,16,32,64 L)of0.05%H2O2solution.Allthereactionsweredetectedat650nmforTMBor415nmforABTSusingthemicroplatereader.Catalyticparametersweredeterminedby ttingtheabsorbancedatatotheMichaelis–MentenequationasEq.(1),whichdescribestherelationshipbetweentheratesofsubstratesconversionbyPB-FtNPsandtheconcentra-tionofthesubstrates.

V=

Vmax S m(1)

Inthisequation,Vistherateofconversion,Vrateofconversion,[S]istheconcentrationmaxisthemaximumofsubstrateandKmistheMichaelisconstant.Thephysical

J.Nanosci.Nanotechnol.12,1–8,2012

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