双液相界面共组装制备可调孔径的磁性介孔二氧化硅球及其应用(2015) - 图文

JournaloftheAmericanChemicalSocietyArticlelargerthanthatofMagn-MSMs-170(5.0nm),andMagn-MSMs-Thesystemwasstirredat170,250,and500rpm,respectively,500hastwosetsofmesoporeslocatedat8.8and18.8nm,andsubsequently,asmallamountofwatersolutionwaswith-respectively.Itsuggeststhattheincreaseofprestirringratehelpsdrawnfordynamiclightscattering(DLS)measurementsat25°C.toexpandthemesoporessize,buttoofaststirringcancauseanAccordingtotheDLSresults,micelleshavearelativelyuniforminhomogeneousporeexpansion.Tofurthergaininformationsizeofabout6.0nmat170rpm(FigureS8a),andwhentheabouttheinteriorstructureofthesamples,theMagn-MSMsprestirring(priortoaddingTEOS)rateisincreasedto250rpm,samplewasslicedforTEMobservation.Themagneticcoreandmostofthemicellesizeincreasesto～20.2nm(FigureS8b),radiallyalignedmesoporeswithauniformsizecanbeclearlyalthoughsmallamountofmicellesremainthesizeofaboutvisibleintheslicedMagn-MSMs-170(FigureS6a).Whileinthe6.0nm.Withafaststirringof500rpm,mostmicellesachieveaslicedMagn-MSMs-500,inadditiontoradialmesoporesofsizeofabout20.0nm;meanwhilesomelargermicellesofaboutabout9.0nm,somelargermesoporesofabout30?40nmcan70.0nm(FigureS8c)arepresentduetothesolubilizationofbeobserved(FigureS6b),ingoodagreementwiththeSEMCTABmicellesenhancedbythedynamicshearingforceofhigh-images(FigureS2c).Table1showsthatthesampleswitha

speedstirring.

Basedontheaboveresults,weproposedashearingassistedTable1.TexturePropertiesandMagneticPropertyofthebiliquidinterfacecoassemblymechanismfortheformationofObtainedMagn-MSMs

core?shell?shellmagneticmesoporoussilicamicrospherestotal(Magn-MSMs).AsdepictedinFigure4,atthebeginning,atwo-phasesystemisformedbyaddinghydrophobicn-hexaneintosamplesareaBET(msurface2/g)a(cmvolumepore3/g)bpore(nm)sizecmagnetization(emu/g)

theaqueoussolutioncontainingCTABsurfactant,FeMagn-MSMs-1706230.915.034.5microspheres,andammonia?water.Then,undermildmechanical3O4@RFMagn-MSMs-2505070.989.0

36.8stirringat170?500rpm,n-hexanecanbecontinuouslyabsorbed196Magn-MSMs-500

498

0.96

8.8,18.8

37.1

bythepreformedCTABmicellesduetothestronghydrophobic50abinteractionbetweenCTABandn-hexanemolecule,whichis5TheBETspeci?csurfaceareaswerecalculatedusingadsorptiondata.srelatedwiththe“likedissolveslike”principle.Alsothewell-cinaj/basedP/PmatchedchainlengthofCTABandn-hexanehelpstostabilize120derivedon0rangefromthevolumeof0.05?theadsorptionadsorbed0.25.bThebranchesatPtotal/Pporevolumeswereestimated0～of0.995.ctheisothermsTheporebysizesusingwerethethecompositemicelles,38,39althoughthedistributionofn-hexane1.0BJHmethod.

inCTABmicellesvariesdynamicallybetweenthewaterand1 :in-hexanebulkphaseduringthecontinuousstirring.Thesizeofod theexpandedcompositemicellesisrelatedwiththeshearing|largermesoporehavelowersurfaceareasandlargerpore 51forceofthemechanicalstirring.Afterthat,upontheadditionof0volumes.2 ,TEOSinton-hexanephase,oligomersilicatenanoclusterscould1～Theyallhavehighsaturationmagnetizationvaluesof3 begeneratedattheoil/waterinterfacethroughhydrolysisandyfor36practicalemu/g,magneticwhichisseparationfavorableapplications.forafastmagneticItisworthresponsenotingluJthat,sincetheRFinterlayercanberemovedbycombustionincondensationofTEOScatalyzedbyammonia?water.They :)bair,suchacore?shell?shellstructurecanbeeasilyconvertedfurtherassociatewithCTABmicellesthroughelectrostaticinter-eWintoauniqueyolk?shellstructurewithmagneticcoretrappedaction,coassembleintorodliken-hexane-CTAB-silicacomposite( etinahollowmesoporoussilicamicrosphereviacalcinationtreat-micelleswithdi?erentsizes,anddepositonFeaDradialalignmentduetotheelectrostatic3O4@RFmicros-pheresthroughainterac- mentinairat500°Cfor5h(FigureS7).

nioFormationMechanism.Theabove-mentionedresultsclearlytionbetweenthecompositemicellesandhydrophilicRFshellandtaciindicatethattheevolutionofthemorphologyandporesizethelowestinterfaceenergyrequirement.23,40AfterthestructurelbofMagn-MSMshasdirectrelationshipwiththeprestirringrate.ofthecompositeis?xedwithacross-linkedsilicaframework,Pu Togaininsightaboutthein?uenceofstirringrate,wepreparedaCTABandn-hexanecanbecompletelyremovedthroughacetonebiliquidsystemofCTAB-ammonia?water?Fe3O4@RF/n-hexane.

treatment,resultingincore?shell?shellmagneticmesoporous

Figure4.SchematicillustrationoftheformationprocessforMagn-MSMswithtunablemesoporesizeviathebiliquidinterfacecoassembly.Inthebiliquidprestirringsystem,atdi?theerentwaterspeedsphaseofconsists170?500ofrpmwater,leadsFe3Oto4@RFCTABmicrospheres,micellesofdibasic?erentcatalyst,sizesandasasphericalresultofCTABthecontinuousmicelles.BeforedynamicaddingsolubilizationTEOS,theofncoassemble-hexaneintotheelectrostaticintoCTABrodlikemicelles.interactioncompositeAfterandthemicellesintroductionlowestwithinterfacediof?erentTEOS,energysizes,therequirement,depositgeneratedonnegativelytheandinterfacecharged?nally?xofsilicaoligomersinteractwiththemicellesinwater,theFemesostructure3O4@RFmicrospheresonFethroughmicrospheres.aradialalignmentThus,afterdueremovingCTABandn-hexane,Magn-MSMswithmesoporoussilicashellofdi?erentporesizescanbegenerated.

3O4@RFE

J.Am.Chem.DOI:Soc.10.1021/jacs.5b05619

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Downloaded by NORTHWESTERN POLYTECHNICAL UNIV on September 11, 2015 | http://pubs.acs.org JournaloftheAmericanChemicalSocietysilicamicrosphereswithdi?erentmesoporessize.Inthewholesynthesis,n-hexaneservesastheoilphasetoexpandthemicellesizesthroughdynamicallyswellingCTABmicelles.

ImmobilizationofTrypsinforSizeExclusionProteolysis.Proteolysisisanimportantbiocatalysisprocess,whichisausefulpathway41,42toacquireinformationonproteinsinproteomeanalysis.Thelowmolecular-weight(MW)proteomeshavearousedgreatinterestinunderstandingpathologicalandbio-medicalsystemsfortheir43potentialapplicationinthebiomarkersandsignalmolecules.However,thelowMWproteomedeterminationsareconsideredtobeagreatchallengeduetotheinterferenceofabundanthighMWproteomes.44,45Here,inthisstudy,weinvestigatedthefeasibilitytosolvetheseproblemsbyusingourMagn-MSMsthatpossesstwoimportantmerits,thatis,thelargeandtunableporesizesforimmobilizationofenzymeforpossiblesize-selectiveproteindigestion,andtheconvenientmagneticseparation.Enzymeimmobilizationhelpstoincreasethestabilityandactivityofenzymeduetoavoidanceofproteinself-enzymolysis,46unfolding?49andaggregationafterimmobilizedonsolidsupports.Traditionalcore?shellstructuralmagneticmesoporoussilicaparticlespossessmesoporesof2nmin196diameter,whichistoosmallforimmobilizationofenzymeinto50thepores.Ourmagneticmesoporoussilicamicrosphereswithb5.swell-controlledmesoporesof5.0?9.0nmcanbeservedasancajidealenzymesupports(Scheme1b).Trypsin,asphericalenzyme/120withsizeof3.8×3.8×3.8nm3,waschosentoimmobilizeinto1.0themesoporesofMagn-MSMs-250.Beforeimmobilization,1 :iGLYMOwasusedasalinkerfortrypsinduetothestrongod interactionofepoxygroupofGLYMOwithfunctionalgroupin| 51aminoacidresiduesoftrypsin.Throughsimpleaqueousreaction,02 trypsincouldbeimmobilizedintothemesoporesofMagn-,13 MSMs-250within1h,whichwascon?rmedbytheUVabsorp-ylutionvalueofthesupernatanttrypsinaftertheimmobilizationJ :)bprocedure.TheimmobilizationcapabilityofMagn-MSMs-250eWfortrypsincanreach97μg/mg(enzyme/supports),higherthan( emostofthereportedresultsbasedonmesoporoussilica.50taDFouriertransforminfrared(FTIR)spectra(FigureS9)ofMagn- nMSMs-250afterimmobilizingtrypsinexhibitnewcharacteristiciotacabsorptionbandsintherangeof1200?1600cm?1,furtherilbcon?rmingasuccessfulimmobilization.

Pu TrypsinimmobilizedMagn-MSMs-250(denotedasMagn-MSMs-trypsin)wasusedforthesizeexclusionproteolysis.Severalsmallproteinswithdi?erentmolecularweightandsizeincludingmyoglobin,lysozyme,andCyt-c49mixedwithalargeproteinBSA,respectively,weretestedforsizeexclusionproteolysisbyusingMagn-MSMs-trypsinwiththemesoporesizeof9.0nmindiameter.Forcomparison,theseproteinpairswiththesameconcentrationof0.5g/Lwerealsodigestedusingequalfreetrypsininaqueoussolutionfor2h.ThepeptidesdigestedfromtheseproteinswereeasilyseparatedwithamagnetanddetectedbyMALDI-TOFmassspectrometry.PeptidesdigestedfrommyoglobinandBSAwerebothdetectedbytheMALDI-TOFusingfreetrypsindigestioninsolution(Figure5a).However,byusingMagn-MSMs-trypsin,onlyasmallamountofpeptidefromBSAwasdetectedandadramaticallyincreasedamountofpeptidesfrommyoglobinwasdetected.ItimpliesthattrypsinimmobilizedinMagn-MSMsisaccessibleformyoglobinandinaccessibleforBSA,demonstratingane?cientsizeexclusionproteolysis.ThesequencecoverageofBSAandmyoglobinbyfreetrypsindigestionturnout30%and18%,respectively.WhileusingimmobilizedtrypsinonMagn-MSMstodigesttheproteins,thesequencecoveragesof5%forBSAand33%formyoglobinwereobtained,re?ectingobvioussuperioritydigestione?ciency

F

ArticleFigure5.MALDI-TOFspectraoftrypticdigestsof(a)myoglobin,(b)lysozyme,immobilizedandon(c)Magn-MSMsCyt-cmixedmicrosphereswithBSAafterandbeingfreetreatedtrypsinbyattrypsin37°Cforc,2h.ThepeptidepeaksatrelativelyhighS/Narelabeledwithm,L,lysozyme,andbwhichCyt-c,areandassignedBSA,respectively.

topeptidesderivedfrommyoglobin,forsmallprotein.Inthecaseoflysozyme?BSAmixture(Figure5b),usingfreetrypsinfordigestion,abundantBSApeptidefragmentsweredetected.Incontrast,Magn-MSMs-trypsindigestedpeptidesarealmostfromlysozyme.Thesequencecoverage(FigureS10)ofBSAdecreasedfrom40%to9%whilethatoflysozymeincreasedfrom27%to46%byMagn-MSMs-trypsindigestion,furtherindicatingthesize-independentdigestion.Besides,Magn-MSMs-trypsinalsorepresentsprioritydigestionforsmallproteinCyt-cthanforlargeBSA(Figure5c).AlltheseresultsshowthatMagn-MSMs-trypsincandigestproteinswithasmallsizeratherthanlargeBSAbecauseproteinssmallerthanthemesoporesizecaneasilyenterintothemesoporesandarein-poredigested,whilelargeproteinsareexcludedoutside.Themagn-MSMs-trypsincanbeeasilyrecycledbyusingamagnetandexhibitswell-retainedcatalyticperformanceafteruseforeighttimes.TherecycledMagn-MSMs-trypsinafteruseforeightcyclespossessesintactporestructureandcore?shellstructure(FigureS11),indicatinga■

goodstructureandperformancestabilityofthisnovelbiocatalyst.

CONCLUSIONS

Insummary,ashearingassistedinterfacecoassemblyinbiliquidphasesystemshasbeendevelopedtosynthesizehighquality

J.Am.Chem.DOI:Soc.10.1021/jacs.5b05619

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Downloaded by NORTHWESTERN POLYTECHNICAL UNIV on September 11, 2015 | http://pubs.acs.org JournaloftheAmericanChemicalSocietyArticlecore?shellmagneticmesoporoussilicamicrospheres(Magn-WeextendourappreciationtotheDeanshipofScienti?cMSMs)withlargetunablesizeandopenporestructure.ResearchatKingSaudUniversityforfundingtheworkthroughThroughtheinterfacecoassemblyofCTABmoleculesandtheresearchgroupProjectNo.RGP-227.WethankProf.silicaoligomersontheFe3O4@RFmicrospheresinn-hexane/waterbiliquidphasesystem,combinedwithmechanicalstirring,highlydispersibleMagn-MSMswithuniformsizes(～600nm),largeandtunableperpendicularmesoporesof5.0?9.0nm,highsurfaceareaupto623m2/g,andlargeporevolumeof■

GuangrongZhouforhelpinTEMcharacterization.

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ArticleJ.Am.Chem.DOI:Soc.10.1021/jacs.5b05619

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