Transmission electron microscopy of unstained hybrid Au nanoparticles capped with PPAA (plasma-poly-allylamine): Structure and electron irradiation effects

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Transmission electron microscopy of unstained hybrid Au nanoparticles capped with

PPAA (plasma-poly-allylamine): Structure and electron irradiation effects

Gontarda,, Lionel C.; Fernández, Asunción ; Dunin-Borkowski, Rafal E. ; Lucas, Stéphane

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Micron

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2014

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Citation for pulished version (HARVARD):

Gontarda, LC, Fernández, A, Dunin-Borkowski, RE & Lucas, S 2014, 'Transmission electron microscopy of

unstained hybrid Au nanoparticles capped with PPAA (plasma-poly-allylamine): Structure and electron irradiation

effects', Micron, VOL. 67, p. 1-9. <http://www.sciencedirect.com/science/article/pii/S0968432814001218#>

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ContentslistsavailableatScienceDirect

Micron

jo u r n al h om ep a g e :w w w . e l s e v i e r . c o m / l o c a t e / m i c r o n

Transmission

electron

microscopy

of

unstained

hybrid

Au

nanoparticles

capped

with

PPAA

(plasma-poly-allylamine):

Structure

and

electron

irradiation

effects

Lionel

C. Gontard

a,∗

_,

_Asunción

_Fernández

a

_,

_Rafal

_E.

_{Dunin-Borkowski}

b

_,

Takeshi

Kasama

c

_,

_Sergio

_{Lozano-Pérez}

d

_,

_Stéphane

_Lucas

e

a_Instituto_de_Ciencia_de_Materiales_de_Sevilla_(CSIC),₄₁₀₉₂_Sevilla,_Spain

b_Ernst_Ruska-Centre_for_Microscopy_and_Spectroscopy_with_Electrons_and_Peter_Grünberg_Institute,_{Forschungszentrum}_Jülich,_D-52425_Jülich,_Germany c_Center_for_Electron_Nanoscopy,_Technical_University_of_Denmark,_DK-2800_Kongens_Lyngby,_Denmark

d_University_of_Oxford,_Department_of_Materials,_Parks_Road,_Oxford_OX1_3PH,_UK

e_NARILIS_–_NAmur_Research_Institute_for_LIfe_Sciences,_Research_Center_in_Physics_of_Matter_and_Radiation_(PMR),_Laboratoire_d’Analyses_par_Réactions Nucléaires(LARN),FUNDPUniversityofNamur,Belgium

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received12May2014

Receivedinrevisedform11June2014 Accepted12June2014

Availableonline20June2014 Keywords:

Poly-allylamine Hybridnanoparticles

Organic–inorganicnanoparticles Transmissionelectronmicroscopy EFTEM

Irradiationeffects

a

b

s

t

r

a

c

t

Hybrid(organic shell–inorganic core) nanoparticleshave important applicationsin nanomedicine. Althoughtheinorganiccomponentsofhybridnanoparticlescanbecharacterizedreadilyusing con-ventionaltransmissionelectronmicroscopy(TEM)techniques,thestructuralandchemicalarrangement oftheorganicmolecularcomponentsremainslargelyunknown.Here,weapplyTEMtothe physico-chemicalcharacterizationofAunanoparticlesthatarecoatedwithplasma-polymerized-allylamine,an organiccompoundwiththeformulaC3H5NH2.Wediscusstheuseofenergy-ﬁlteredTEMinthe low-energy-lossrangeasacontrastenhancementmechanismforimagingtheorganicshellsofsuchparticles. Wealsostudyelectron-beam-inducedcrystallizationandamorphizationoftheshellsandtheformation ofgraphitic-likelayersthatcontainbothCandN.Theresistanceofthesamplestoirradiationby high-energyelectrons,whichisrelevantforopticaltuningandforunderstandingthedegreetowhichsuch hybridnanostructuresarestableinthepresenceofbiomedicalradiation,isalsodiscussed.

1. Introduction

In the past decade, the study of hybrid nanoparticles(NPs) hasbecomeamajorﬁeldofinvestigationincolloidand materi-alsscienceandhasledtoavarietyofapplications,especiallyin nanomedicine(Bawarski et al.,2008).Hybridorganic–inorganic NPs that are capped with polymers and naturally occurring biomolecules areof great interest for targeting vascular, extra-cellular and cell surface receptors. Although the inorganic components of hybrid NPs can be characterized readily using conventionaltransmissionelectronmicroscopy(TEM)techniques, a knowledge of the structural arrangement of the surrounding organiccomponents,whichisrequiredtoestablishabetter under-standingand controlover NPsynthesisand properties,remains largelyunknown.Thissituationresults,inpart,fromthefactthat

∗ Correspondingauthor.

E-mailaddress:lionel.cervera@icmse.csic.es(L.C.Gontard).

softmaterialshavepoorelectron-opticalimagecontrastandare sensitiveto the ionizing radiation that is used in conventional TEMs(Egertonetal.,2004).Theuseofheavy-atomstainingisan alternativeapproach (Chenetal., 2006), but itdegradesspatial resolutionandisdifﬁculttocontrol,whileelectron-beam-induced effects,suchasheating,hydrocarboncontaminationandcharging, canaffectthestructuresandstabilities ofsuchsamples(Martin et al., 2005;Libera and Egerton, 2010). Toan extent, electron-beam-inducedheatingandcontaminationcanbereducedbyusing cryogenic stages, while chargingcan bereduced by using con-ductive coatings.Insomecases,core–polymeric–shellNPshave beencharacterizedsuccessfullyusingTEM(KangandTaton,2005; Liet al.,2009)and high-angleannulardark-ﬁeld scanningTEM (HAADFSTEM)(Krivaneketal.,2010;vanSchooneveldetal.,2010), butingeneralonemustattempttodelaytheonsetofdamageby usinglow-dosetechniques(Malacetal.,2007),lowaccelerating voltages(Drummyetal.,2004),lowspecimentemperatures(Libera andEgerton,2010)and,morerecently,directelectrondetection (Gontardetal.,2014a).AlthoughphaseplatesforTEM,including moreunusualsuggestionssuchaslaser-basedphaseplatesbased

http://dx.doi.org/10.1016/j.micron.2014.06.004

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2 L.C.Gontardetal./Micron67(2014)1–9

ontheKapitza–Diraceffect(Mülleretal.,2010), mayprovide a solutiontosomeoftheseproblemsinthefuture,these technolo-giesare currentlyeitherunderdevelopmentor stillimpractical forcontinuoususe(NagayamaandDanev,2008).Nevertheless,in conventionalelectronmicroscopes,damageofelectrically insulat-ingpolymersisunavoidable,inpartbecausefreeelectronsinthe specimencannotcompensateforradiation-inducedfreeradicals onatimescalethatisshorterthanthatrequiredforotherchemical processestooccur.

Here,we assess theapplication of differentTEMtechniques forthecharacterizationof AuNPsthat haveamino functionali-ties.TheNPsaresynthesizedusingplasmavapordepositionofAu (cores)andallylamine(caps),resultingintheformationof plasma-polymerized-allylamineshells.Suchcappednanoconjugateshave beensuccessfullycovalentlyimmobilizedviaanamidelinkageto Cetuximabmonoclonalantibodies(Maregaet al.,2012).Invivo studiesofthenanoconjugatesdemonstratedactivetumor target-ing,openingnewpossibilitiesforcancertreatment(Karmanietal., 2013).

Polymers (including plasma-poly-allylamine (PPAA)) consist largelyoflightelements, whoseelasticinteractionswithhighly energeticelectronsarerelatively weak,while inelastic (energy-loss)processes arerelativelystrong(Liberaand Egerton,2010). Therelativelyhighfractionofinelasticallyscatteredelectronsfor lighter elementsin polymers providesan alternative source of contrast,by using electron energy-loss spectroscopy (EELS) for mappinglocal changes in composition and the distributions of phaseswithouttheneedtouseheavy-elementstains.The versa-tilityofthistechnique,whichhasbeenusedtostudymultiphase polymermorphologyinblends,compositesandblockcopolymers, resultsfromthefactthatincidenthigh-energyelectronscanexcite valence electrons, inter- or intra-band transitions or plasmons (collectivelongitudinaloscillationsofvalenceorconductionband electrons).Inaromaticpolymers,acharacteristicenergy-losspeak at_∼7eVhasbeenassignedtoanelectronic_␲–␲*transition(Liand Egerton,2004).However,itcanalsoresultfromelectronirradiation duetohydrogenabstractionfollowedbyareactionbetween adja-centprimary-chaincarbonatoms.SurfaceplasmonsinNPs,which featureinthesub-10-eV(optical)regionsofEELspectra,havebeen studiedintensivelyasaresultoftheirimportanceinplasmonics andbiomedicine(Jainetal.,2007;Haridasetal.,2008).In con-trast,therehasnotbeenanequivalenttheoreticaleffortaimedat understandingbulkplasmons,whichtypicallyhaveenergiesabove 10eV.

Theselectionofinelasticallyscatteredelectronsthatliewithin anarrowenergyrangeformsthebasisofcompositionalmapping usingeitherspectrum imagingor energy-filteredTEM(EFTEM). EFTEM core-loss imaging of C, N and O has been invaluable forprovidingmicrochemicalandtopochemicalinformationabout polymerfilmsandcomposites(DuChesne,1999).However,forNPs, damageandcontaminationofthespecimenbytheelectronbeam resultsinuncertaintyintheanalysisofthesignalsandinaloss ofresolution.Recentdevelopmentsincludetheuseofdedicated insituplasmacleaningintheTEMforEFTEMCK-edgestudiesof hybridAu@polymerspecimens(Horiuchietal.,2009).Although low-lossEFTEMinthesurfaceplasmonrange(<5eV)isnowused intensively(e.g.,Nelayahetal.,2009)thebulkplasmonrangeis muchlessexploredbecauseofthedifficultyofdatainterpretation (Howie,2003).Howeverbulkplasmonscanbebeenusedtocreate high-contrastimageswithhighsignal-to-noiseratiostodistinguish betweendifferentcarbonaceousmaterials(Huntetal.,1995;Du Chesne,1999;Danielsetal.,2003;Linaresetal.,2009).

Here,weuseaberration-correctedTEMandthinsample sup-portsatroomtemperaturetorecordbothlow-lossandcore-loss EFTEMimagesofunstainedAuNPsthat arecoated withPPAA, anorganiccompoundwiththeformulaC3H5NH2(Moreauetal.,

2009).ThesizesoftheAuparticlecoresrangebetween1and10nm, while the thicknessesof the organicshells are typically below 2nm.Weshowthatitispossibletoobtainhighspatialresolution physico-chemicalinformationabouttheorganicshellsanddiscuss theapplicabilityofbulk-plasmonEFTEMforimagingtheshellswith highcontrast.Wealsodiscusstheeffectsofelectron-beam-induced degradationinthecontextoftheeffectsofbiomedicalradiation techniquesonsuchNPs.

2. Methods

2.1. Specimenpreparation

Several methods of capping Au NPs with PPAA have been devised,includingaqueous-phasetechniques(Sardaretal.,2007) and layer-by-layer deposition of polyelectrolytes (Gittins and Caruso,2001;Masereeletal.,2011).Here,weusedagas-phase physicalprocessthatprovidesamethodfor NPproductionthat isindependentofthesubstrate.Inordertoproducelarge quan-titiesofmaterial,werepeatedthedepositionofseveralstacksof NaCl/Au/PPAAonastainlesssteelsubstrate(3in.).After deposit-ing more than 30 of these stacks sequentially, the substrate wasimmersedinwaterin ordertotransferthecapped AuNPs (Au@PPAA)intosolutionthankstotheNaCldissolution(Moreau etal.,2009).PPAAcappingpreventsaggregationandprecipitation andissuitableformABlabeling.

Theprocedurefortheproductionofeachstackwasasfollows. WeusedaPVDvacuumsystemfromAJAInternationalequipped withtwomagnetronsputteringsources(bottomupconﬁguration). Thechamberwaspumpeddownto10-4_Pa_prior _to_the_process

(turbomolecularpump).Thespecimenholderwaselectrically iso-latedfromthechamber,andconnectedtoaradiofrequency(RF) powersupply(13.56MHz).Goldnanoparticleswereproducedby usingonemagnetrongunequippedwitha99.99%goldtarget(2in. diameter),poweredinDCmodeat75Wandat24Pafor2s.This highpressureensuresgoldNPformation.Theﬁnalmonolayer(ML) equivalentthicknesswas0.5nm.Once thesample wascovered withAuNPs,PPAAfunctionalizationtookplacethankstoanRF ally-lamineplasmaimpingingonthesubstrateholderat10WRF,8Pa for20s.ThePPAAthicknesswasabout1nm.Thesamplewasthen movedabovethesecondmagnetrongunequippedwithastainless steelcrucible(2in.diameter)ﬁlledwithNaCl(Merck).NaCl depo-sitionwascarriedoutat30WRF,13Pafor60sforatotalthickness of30nm.Theprocedurewasthenrepeatedabout30times:the AJAsystemisfullyautomatedanda recipewassetuptoensure repeatabilityfromproductiontoproduction.

2.2. Characterization

ForTEMobservation,adropofthesolutionwasdepositedonto athinCsupportfilmonaCuTEMgridanddriedinair.The sup-portingC filmsusedin thepresentexperiments wereobtained fromTedPellaandhadthicknessesofbelow10nm.Bright-field andhigh-resolutionTEMimageswereacquiredat300kVwithan aberration-correctedFEITitanfieldemissiongun(FEG)TEMusing a2048×2048pixelcharge-coupled-device(CCD)camerawithout binningandatypicalacquisitiontimeof2s.EFTEMimageswere acquiredusingaGatanimagingfilter(GIF)thathadameasured energyresolution (fullwidth athalfmaximum ofthezero-loss peak) of ∼1.2eV.1024×1024 pixel EFTEMimages in the bulk plasmonrangewereacquiredusing2×hardwarebinning,a4eV energy-selectingslit,anacquisitiontimeof7sandanedgeonset energyof 27eV.Core-loss EFTEMwasusedtoidentify elemen-taldistributionsofNusingthethree-windowsmethod,whereby images are recorded using two pre-edge and one post-edge

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window.Thepre-edgeimagesareusedtosubtractthebackground inthepost-edgewindow,assumingapowerlawbackgroundofthe formAE−r,whereAandrareﬁttingparametersandEistheenergy loss.512×512pixelN core-losselementalmapswereacquired using4×hardwarebinning,a30eVenergy-selectingslit, acqui-sitiontimesof10s,edgeonset energiesof353,383and416eV andthree-windowbackgroundsubtractioninDigitalMicrograph softwarefromGatan.EELspectrawereacquiredbyoperatingthe TEMinspectroscopymode.Adispersionof0.5eV/channelwasused andthespectrawereprocessedusingDigitalMicrographsoftware fromGatan.Energy-dispersiveX-rayspectroscopy(EDXS)was per-formedat300kV usingaTecnai F30FEGTEMequippedwitha windowlessX-MAXSidriftdetectorfromOxfordInstrumentswith adispersionof5eV/channelanda6␮spulseprocessingtime.The samplewastiltedby30◦ andspectrawereprocessedusingTIA softwarefromFEICompany.

Atomisticmodelsofcore–shellNPsweregeneratedusinga ded-icatedsoftwareprogramwritteninMatlab.Theshellwasmodeled bystartingfromahypotheticalC3Ncrystal(withthecorrectC/N

ratioforPPAA)comprisingafacecenteredcubicstructurewithone Catomateachcorneroftheunitcellanda Natomatthe cen-terofeachface.Theunitcelldimensionwaschosentobe0.2nm, sothat theaveragedistancebetweentheC andNatoms corre-spondstothetypicalbondlengthexpectedinamines(∼0.148nm). Inordertomodelanamorphousshell,thecrystallinepositionof eachatomwaschangedfromitsstartingpositionbyarandom num-bermultipliedby20%oftheunitcelldimension.Thismodelwas reﬁnedbydeletingouteratomsselectivelyuntiltheshapeofthe shellresembledthatobservedinexperimentalimages.Aholewas thencreatedinthecenteroftheshelltoaccommodateaAuNP. High-resolutionTEMimagesweresimulatedusingthemultislice algorithminJEMSsoftware,withthetransferfunctionofthe micro-scope(defocus+90nm;defocusspread20nm;beamconvergence semi-angle2mrad)chosentoprovideamatchtothe experimen-tallyobservedcontrastand 1%randomnoiseaddedtotheﬁnal simulations.Thelargedefocususedintheexperimentalimagesand

simulationsisareﬂectionofthefactthatcontrastenhancementof theshellsaroundtheNPsisneeded.

Casinov3.2softwarewasusedtoperformMonte Carlo sim-ulations of inelastic electron scattering events and trajectories (Demersetal.,2011).Inthesimulations,whichwereperformed with100electronsandparallelillumination,spherical5nmAuNPs weresemi-buriedin10nm3_of_amorphous_C.

3. Results

Fig.1ashowsa conventionalBFTEMimageoftheAu@PPAA NPssupportedonultrathinC.Theﬁeldofviewis300nmandthe darkparticlesofdifferentsizecorrespondtotheAucores.Shells arevisiblearoundthelargerparticles.Thetwo mainsourcesof contrastinBFTEMimagesaremass-thicknessanddiffraction con-trast.Theshellshaveuniformintensityirrespectiveoftheirsize, suggestingthattheyareamorphous.Fig.1b–dshows representa-tiveresultsofchemicalanalysisperformedbyaveragingEDXSand EELSsignalsoveranareaofthespecimensimilartothatshown inFig.1a.TheEDXspectruminFig.1aconﬁrmsthepresenceof NbutalsoindicatesthepresenceofSi.Thelatterpeakmaybean artifactoriginatingfromthewindowlessSidetector.Fig.1cshows anEELspectrumofthecore-lossNK-edge(401eV)andOK-edge (532eV),whileFig.1dshowsacorrespondingspectrumoftheC K-edge(284eV).TheNpeakistheprimarysignatureofthepresence ofallylamine(C3H5NH2)inthespecimen.Althoughpristine

ally-laminecontainssingleC Nbonds,sharpNandOedgeonsetsmay indicatethepresenceofC OandC Ngroups(GarvieandBuseck, 2004).Suchgroupsmayformduringtheplasmadischargewhen theprecursor(allylamine)isdissociatedintofragmentsofdifferent masscontainingC C,C N,C HandN Hbonds.Thebreakingof N HbondscanleadtotheformationofC Ngroupsduring recom-bination.PPAAcoatingsmayalsooxidizeinambientair,resulting intheformationofC OandC Obondsduetopolymeroxidation (Masseyetal.,2010).

Fig.1. (a)Bright-ﬁeldTEMimageofhybridAunanoparticlescappedwithallylaminesupportedonultrathin(approx.10nm)amorphousCacquiredafteranelectrondose exposureof250C/cm2_._The_Au_cores_(darker)_are_surrounded_by_shells_of_lower_density_(lighter)._The_shells_do_not_enclose_larger_particles_completely._(b)_EDX_spectrum showingpeaksofN,AuandasmallamountofSi.(c)Core-lossEELspectrumshowingNandOK-edges.(d)Core-lossEELspectrumshowingtheCK-edge.ThestrongCu peaksoriginatefromthegridbarsoftheTEMCugrid.

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TheC K-edgeinFig.1dissimilartothatexpectedfor amor-phousC,withthelossofstructuralorderresultinginarelaxation oftheselectionrulesforthe1sto␴*transitionandproducinga broadfeaturelesspeakat290–310eV.Thespectrumcontainstwo strongpeaksat288.5and300eV,correspondingtoC C␲*and C C␴*bonds(GarvieandBuseck,2004).The␲*to␴*ratiocanbe calculatedbyassigningenergylossesof282–291eVto␲*states andenergylossesof294–301eVto␴*_states_in_the

background-subtractedCK-edgespectrumshowninFig.1d.Weobtainavalue forthe␲*/␴*ratioof0.4,suggesting alackoflong-rangeorder (Katrinaketal.,1992).Thecontributiontothesignalfromthe amor-phousCsupportﬁlmmayaccountforthedominantsignatureof ␴*_type_bonds._On_the_other_hand,_C _C_␲*_peaks_can_indicate_the

presenceof aromaticringsandunsaturated bondsof hydrocar-bons(contamination)typicallypresentinthecarbonﬁlmsusedto supportTEMsamples.Also,C C␲*_peaks_can_originate_from

unsat-uratedbondsthatremaininthePPAAdeposition,asC Cbonds arepresentinallylamine(CH2 CH CH2 NH2);correspondingto

incompletepolymerizationoftheallylamine.Therefore,itis nec-essarytouseanalternativeapproachtodeterminetheoriginofthe C C␲*signal,forexampleusinglow-lossEFTEMinordertomap thecharacteristicenergy-losspeakof_∼7eVthathasbeenassigned toanelectronic␲–␲*transitionofaromaticCrings(LiandEgerton, 2004).

Fig.2ashowsrepresentativeexamplesofhigh-resolutionTEM imagesofAu@PPAANPswithcoresofdifferentsize.Extended elec-tronirradiationresultedintearingoftheCsupportﬁlm,permitting clearvisualizationofNPshellsforcoresizesdownto1nm.The shellstypicallyhavethicknessesof1–2nmanddonotenclosethe NPsuniformly.Fig.2bshowstheparticlesizedistribution(PSD) measuredfrom216NPsbyapplyingasemi-automated segmenta-tionalgorithm(Gontardetal.,2011)totheimageshowninFig.1. ThePSDindicatesthattheAucoreshavesizesofbetween0.9and 8nm,withanaveragevalueof2.25nmandastandarddeviationof 1.09nm.

Fig.2cshowsahigh-resolutionTEMimageofahybridNPwith acoresizeof1.5nmattachedtotheedgeoftheCsupport.Fig.2d showsasimulatedhigh-resolutionTEMimageobtainedfromthe atomisticmodelshowninFig.2e,whichhasa1.5nmAucoreand anamorphousshellmadeof75%Cand25%N.Thecontrastinthe simulatedimagematchesthatintheexperimentalimage qualita-tively.ThereplacementofNatomsinthemodelbyCatomsdidnot resultinasubstantialchangeinthesimulatedimage,conﬁrming thelackofsensitivityofhigh-resolutionTEMtochemicalchanges forsmalldifferencesinatomicnumber(Z=6forCandZ=7forN). ThebrightrimaroundtheAucoreinthesimulationinFig.2dis associatedwiththefactthatinthemodelthereisagapbetween thecoreandtheshell.Theabsenceofsuchabrightriminthe exper-imentalimagesuggeststhatthecoreiscappedcoherentlybythe PPAA.Thebrightrimonthesurfaceoftheshellisconsistentwith thedefocusvalueof+90nmusedinthesimulation.

Fig.3ashowsalow-lossEFTEMimageacquiredfromthe spec-imenshown inFig.1atanenergy lossof27eV,which isclose tothebulk plasmonenergyexpectedfor Cthatcontains short-range(graphitic)order.Fig.3bandcshowszero-lossandlow-loss images,respectively, oftwo interactingparticlesindicated by a whitesquareinFig.3a.Fig.3dshowsaratioofthetwoimages. Thegraylevelsprovideameasureoftheplasmonexcitation prob-abilityatagivenpositionintheimageintegratedoverthewidthof theenergywindow(Stöcklietal.,2000).Theratioimageindicates thattheplasmonresonancesareparticularlyintenseattheAucore, theAu–shellinterfaceandtheoutersurfaceoftheshell.

Fig.4illustratestheeffectoftheelectronbeamonthespecimen. During TEM observation, the C support ﬁlm thinned progres-sivelyuntilholesformed.SomeNPsthatwereclosetoeachother coalescedorchangedinshape,whiletheshellsaroundtheNPs

Fig.2.(a)Examplesofhigh-resolutionTEMimagesofAu@PPAAnanoparticleswith coresofdifferentsizesupportedonthinC.(b)Measuredparticlesizedistribution oftheAucores(mean=2.25nmandSD=1.09nm).(c)High-resolutionTEMimage ofahybridnanoparticlewitha1.5nmcoreattachedtotheedgeoftheCsupport. (d)Simulatedhigh-resolutionTEMimageintensitycalculatedusingtheatomistic modelshownin(e).Themodelhasa1.5nmAucoreandanamorphousshellthat contains75%Cand25%N.

changeddramatically.Fig.4aandthecorrespondingdiffractograms below it show theevolution of two large NPs(numbered 5 in

Fig.5),whichfusedintooneparticleandbecameenclosedbya commonshell.ThefirstimageinthesequenceshowstwoAuNPs thatareincontactandsurroundedbyanamorphousPPAAlayer. Thesecondimageshowsthattheparticlesstarttocoalesceand thata commonshellis createdaround bothparticles.Theshell isnowcrystallinewith∼6independentgraphene-likelayerswith twodifferentinterplanarspacingsvisiblein twodirections.The firstspacingof0.33nmissimilartothec-axisspacing between graphene sheets in graphite of 0.34nm. The second spacing of 0.37nmissimilartothatreportedforgraphene/Cshells surround-ingAuNPssynthesizedusingchemicalvapordeposition(Chopra etal.,2009).Inthethirdimage,thetwoNPshavefusedtogether andtheshellisnowdamaged.ItisremarkablethattheNPswere able tocoalesceunder intense electron beamirradiation while retainingtheshellaroundthem.Inarelatedexperiment,Auhas beenobservedtoflowbetweendefectivegrapheneparticles(Sun etal.,2008).TheimagesequencesinFig.4billustratethesame

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Fig.3.(a)EFTEMimageacquiredinthelow-energy-lossrangeforbulkplasmons usinga4eVenergy-selectingslitcenteredonanenergylossof27eV,whichisclose tothebulkplasmonenergyforCwithshort-range(graphitic)orderandalsoof Au(∼25eV).(b)Zero-lossimageofadimerofinteractingAuNPsindicatedwitha whitesquarein(a).(c)Low-lossimageoftheparticlesshownin(b).(d)Ratioof image(c)dividedbyimage(b).Thegraylevelsin(d)provideadirectmeasureofthe plasmonexcitationprobabilityatagivenpositionintheimageintegratedoverthe energywindow.Theplasmonresonancesareparticularlyintenseintheparticles, attheAu–shellinterface,inthecontactregionbetweentheAuparticlesandatthe outermostsurfaceoftheshell.

irradiation-inducedtransitioninotherparticles (numbered3in

Fig.5).Fig.4c–eshows simulationsof theexpectednumber of inelasticevents(showninpink)generatedbytheimpactof100 electronsacceleratedby60V,20kVand300kV,respectively.For 60V,theyieldis1andtheinelasticeventsremainatthesurface. For20kV,theyielddecreasesbutthe“damage”affectstheentire volumeofthespecimen.For300kV,thenumberofinelasticevents issmallerthanfor20kV.

InordertoconﬁrmthattheorganicshellisPPAA,weusedEFTEM tomeasurethepresenceandlocaldistributionofN,whichisa con-stituentofallylamine,withhighspatialresolution.Imageswere acquiredatatimewhenthePPAAcoatinghadalreadystartedtobe damagedbytheelectronbeam.Fig.5ashowsazero-lossEFTEM imagerecorded from“area1”indicated in Fig.1.Fig. 5band c showscorelossEFTEMimagesacquiredattheNK-edge(401eV). Althoughspecimendriftand/ordamagewillhaveaffectedpartsof thebackground-subtractedelementalmapsshowninFig.5bandc, theresultsarestronglysuggestiveofthepresenceofNaroundthe AuNPs.

4. Discussion

We ﬁrstdiscussthepossibleoriginof thehighcontrast fea-turesinthelow-lossEFTEMimagesshowninFig.3.Second,we analyzetheeffectsofelectronirradiationontheintegrityofthe specimen.Third,weassessthephasetransformationsthatoccur afterprolongedelectronirradiation,includingcrystallizationand amorphization.

4.1. EFTEMcontrast:bulkorsurfaceplasmons?

InFig.3,thehighrecordedintensityattheAu–PPAAinterface andattheoutermostsurfaceofthecappinglayerislikelytobe associatedwiththeexcitationofplasmonsintheshelland/orthe Aucore.Theimageswererecordedatanenergylossof27eV.For aAuNP,thebulkplasmonenergyistypically∼25eV,althoughthe precisevaluecandependonthenatureofthesurfaceandthe oxi-dationstate(Tsivadzeetal.,2013).Surfaceplasmonscanalsobe excitedattheAucore,buttheyaretypicallyobservedintheoptical rangenear530nmor2.3eV.Boththeoreticallyand experimen-tally,shiftsofsurfaceplasmonenergiesinNPshavebeenshownto dependontheelectronicpropertiesoftheparticles.However,such energyshiftsaremostlyblue-shiftsandtheirmagnitudesrarely exceed10eV(Jainetal.,2007;Haridasetal.,2008).Hence,ashift ofthesurfaceplasmonenergyoftheAucoreisnotlikelytobethe sourceofthestrongintensityintheshellsobservedinFig.3.

ThebulkplasmonenergyforCisbetween22and27eV, depend-ing on the degree of short-range order present. For graphitic structures,theelectronicpropertiesandhencetheplasmon ener-giesindirectionsparallelandperpendiculartothegraphiticshells aredifferent(Stöcklietal.,2000).Insomecases,surfaceplasmon excitationprobabilitiesfornanotubeshaveresultedin the exci-tationof higher ordersurface plasmonresonance modesabove 10eVduetotheﬁnitelengthsofthenanostructures,whichimpose alowerboundonthewavevectortransferoftheelectronsthat excitestheplasmons(Stöcklietal.,2000).

Giventhedifferentpossiblecontributionstothestrongcontrast observedinFig.3,weareforcedtoconcludethatwedonotyet haveaclearinterpretationofitsorigin,whichislikelytoinclude theroleofinterfacesandelectriccharge.TheenergylossesinFig.3

areintheexpectedrangeforbulkplasmonexcitationsinAuand C,buttheirspatialdistributionisnotdistributeduniformly, sug-gesting thatthedetailedmorphologyofthecore-shelldimerin

Fig.3bandthestructure androleofthesupportmustbetaken intoaccountfor afullinterpretationofthecontrast(Wangand Cowley,1987).Oscillationsofsurfacechargesdependsensitively onthedielectricpropertiesofthematerialand,moreimportantly, onthegeometricalconfigurationoftheinterfaces.Forexample, energy lossesof 20eVhave beenmeasuredusingX-ray photo-electronspectroscopyatthesurfacesofamorphousCfilmswith superficial“polymeric-like”monolayergrafting(Godetetal.,2009). Thissignalwasunderstoodtooriginatefromsurfaceplasmonsin theClayer,ratherthanfrombulklossesinthe“polymeric”grafting. Similarly,thehigherprobabilityofenergylossesfoundatthe inter-facesoftheparticlesinFig.3maybeassociatedwithbulkplasmons originatingfromthecappinglayer.

4.2. Electronirradiationdamage

ThesupportingCﬁlmthinneddownandthePPAAshell amor-phizedanddamagedafterlongexposuretotheelectronbeam.At theacceleratingvoltageusedinthepresentexperiments,knock-on damage,i.e.,thedisplacementofanatomfromitsoriginalsitebythe impactoftheenergeticelectronbeam,isinevitable.Ofparticular relevanceisthelossofatomsthatareexpelledintovacuum (sput-tered)fromthespecimenexitsurface.Theprobabilityofknock-on

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Fig.4.Illustrationoftheeffectoftheelectronbeamonthespecimen.AfterTEMobservation,theCsupportﬁlmthinneddownandsubsequentlyholesopenedacrossit. Theprocessstartedslowlyandthenaccelerated.Someparticlesthatwereclosetoeachothercoalescedorchangedshape,whiletheshellssurroundingtheNPsunderwent dramatictransformations.(a)High-resolutionTEMimagesandcorrespondingdiffractogramsshowingtheevolutionoftwolargeparticlesthatfusedintooneparticleand becameenclosedbyacommongraphiticshell.(b)Exampleofthetransformationofashellintoacrystallineform.(c–e)MonteCarlosimulationsofinelasticinteractions (pinkspots)generatedby60eV,20keVand300keVelectronsirradiatinga5nmAunanoparticlesemi-buriedinamorphousC.(Forinterpretationofthereferencestocolor inthisﬁgurelegend,thereaderisreferredtothewebversionofthearticle.)

damagedecreaseswithincreasingatomicnumberandistherefore lesslikelyforAuthanforH,C,OorN.Despitethenoticeable trans-formationofthehybridNPs(Fig.4),wemeasuredthepresence ofNintheirshellsafter2minofelectronirradiation(Fig.5)even aftertheshellbecameamorphous.Justascoatingoneorboth sur-facesofaspecimenwithCcanhelptopreserveitscrystallinityand toreducemassloss,thepresenceofaC supportﬁlmmayhave preventedsputteringoflightatomsorothermoleculesfromthe organicshellsintovacuum(Egertonetal.,2004).Oneproposed explanationisthatareturntotheoriginalmolecularstate (heal-ingofthebrokenbond)ofanorganicmoleculeismorelikelyifthe escapeofvolatileelementsisprevented(FryerandHolland,1983). Anotherimportantsourceofdamageinorganiccompoundsis inelasticdamagecausedbyvalenceelectron(ratherthancore-loss) excitation. Even relatively radiation-resistant organic materials mayundergosomeformofdamagewhenexaminedinanelectron microscopeatenergiesbelowtheknock-onthresholdenergy.It isinterestingtonotethatholesfrequentlyformedclosetoNPs. ThesimulationinFig.4eindicatesthatat300keVinelasticevents aremore likely tohappenin theAuNPsthan inC. Hence,the

breakingofC CbondsbyradiolysismaysoftentheCﬁlmnearthe particles,enhancingthesputteringrateofCatoms.Anotherform ofradiation-induceddamageisthroughheatdissipationfollowing energy transferin inelasticevents. Also, theelectronbeamcan breakhydrocarbonresiduesandtypicallyresultinapolymerization processthatcanfrequentlycoverareasofthesampleunderstudy, degrading thequality of data acquired.Although the electron-beam-inducedriseinspecimentemperatureisexpectedtobelow fortheexperimentalconditionsusedhere(LiandEgerton,2004), moleculardynamicssimulationshaveshownthatholeformation inthin C ﬁlmscanoccurwhen theelectron beamcausessmall clustersofamorphousC(e.g.,fromhydrocarboncontamination)to undergothermalexplosions(Börrnertetal.,2012).

A more profoundconsequence of inelasticscattering is that it leads to chemical changes in the specimen induced by the generation of low-energy (secondary) electrons. The specimen wasirradiated witha highcurrent ﬂux(25A/cm2_), _well_above

the critical electron dose for damage of PPAA, approximately 0.9×10−2C/cm2_, _based _on _its _melting _temperature, ₍₂₁₆◦_C)

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Fig.5. (a)Zero-lossEFTEMimageof“Area1”indicatedinFig.1.After2minofirradiationwith200keVelectrons(totaldose=3×103_C/cm2_),_the_C_ﬁlm_became_noticeably thinnerandholesformed.(b)Background-subtractedNEFTEMelementalmap.(c)Detailsofzero-lossimagesandNmapsofthreeparticlesin(a)and(b).Althoughspecimen driftand/orspecimendamagemayhaveaffectedpartsoftheelementalmapsshownin(b)and(c),theresultsaresuggestiveofthepresenceofNassociatedwiththepositions oftheAuparticles.

Figs. 4and 5 wereacquired afterexposures of 2min,resulting in a total dose of3×103_C/cm2_. _Low-energy _electrons_that _are

produced by irradiation with high-energy particles may cause primary chain breakage and other molecularfragmentation, as wellastheformationofdoublebondsandcross-linking(Sanche, 2002;Waskeetal.,2012).Secondaryelectronshavelowenergies (usuallybelow70eV)andthermalizationdistancesontheorder of nm, which deﬁne theinitial volumesfor energy deposition. ConsideringthestrongergenerationofsecondaryelectronsinAu thaninC,apolymer-metalinterfaceofahybridNPislikelytobe veryvulnerabletoradiationdamage.ExperimentsonPPAAhave demonstratedthataminegroups(CN)remainstableduring irra-diationwithlow-energy(1–60eV)electronsatlowdosesandthat theprimarychemicalmodiﬁcationsarealossofHandO(surface deoxidation)(Masseyetal.,2010).Emissionthresholdsforcations andanionsinirradiatedPPAA werefoundtobebetween7and 25eV,whichiswithintherangeofenergiesofsecondaryelectrons and collective-excitations (inter band, intra bandand plasmon resonances)generatedbyhigh-energyelectronirradiation(Liand Egerton,2004;Inadaetal.,2011).Theelectron-induced decompo-sitionofadsorbedorganiclayerswithamine(NH2)groupsonAu

substrateshasbeenobservedtobeinitiatedbysecondaryelectrons, transformingthelayerintoanamorphouscarbonnitridethinﬁlm (Wnuketal.,2009).MonteCarlosimulations(seeFig.4)showthat for60eVincidentelectronstheyieldofinelasticeventsis1andthat theyoccurmainlyatthesurfaceofthesample.Inourexperiments,

irradiationwasperformedusing300keVelectrons,whichhavea much higherpenetrationdepth(see Fig.4),generatinginelastic eventsinside thevolume of the specimen and not only onits surface. For our experimental specimenthickness and incident electron energy,we calculated a yieldfor inelasticcollisionsof 5% of the number of incoming electrons. Even after applying a correctionfor the yield, the high current ﬂuxes used in our experimentswereseveralordersofmagnitudehigherthanthose usedbyMasseyetal.(2010),indicatingthatthegenerationofa largenumberofsecondaryelectronsislikelytohavecontributed todamageofthePPAAshellsofourNPs.

4.3. Electron-irradiation-inducedcrystallization

Figs.4and5showaphasetransitionsufferedbytheorganic cappingoftheAu@PPAANPs,ﬁrstbyare-orderingofthelayers andthenbyamorphizationandsubsequentdamage.Inparticular, theformationofgraphite-likelayerswasobserved.AlthoughAuis anoblemetalthatistypicallynotabletocatalyzetheformationof C-basedmaterials,thegrowthofCshellsinthevicinityofAuNPs hasbeenobservedinsituintheTEM.Graphenefragmentsand lay-ersformed,suggestingthatacrystallineAusurfacecancatalyzeand provideatemplatefortheorderingandcrystallizationofC. More-over,underintenseelectronirradiation(50A/cm2_),_the_graphene

fragmentscancloseandformregularsymmetriclayersaroundthe NPsuntilcompleteshellclosureisachieved(Sutteretal.,2005).

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However,thelatterexperimentswerecarriedoutataspecimen temperatureof 550◦C, whiletheexperimentsshown herewere carriedoutatroomtemperature.

Electron-induced graphitization of C is a well-known phe-nomenon, arising from cross-linking between neighboring moleculesatlowtemperature.Asaresultofdifferent hybridiza-tion,Cisabletoformdifferentallotropes,ofwhichgraphiteand diamondarethemostwellknown(Banhart,1999;Shakerzadeh et al.,2012).In particular, high-energy electronirradiation can graphitizefree-standingamorphousCandformConions (spher-icalstructureswithnodanglingbondsthathaveauniformstrain distribution)(Börrnertetal.,2012).Thegraphitization(formation ofC Cbonds)inpolystereneandPPAAasaresultofirradiation withlow-energyelectronshasbeenobserved(Masseyetal.,2010; Leeetal.,2008),inadditiontothelossofOandH.Afterprolonged irradiation,weobservedthatthePPAAshellsweredamaged,but westillmeasuredthepresenceofNusingEFTEM.Therefore,we canconcludethataftercrystallizationC and Nremainedinthe shellsoftheNPsinourspecimen.ThesubstitutionofCbyNin graphiteinaregularfashionmayhaveresultedintheformation of a “carbonnitride” (e.g., C3N4, C3N2, C3N, C5N or C10N3), or

evengraphiticg-C3N4(Thomasetal.,2008).Subsequent

electron-beam-induced decomposition may therefore have transformed thelayerintoanamorphouscarbonnitride,aspreviouslyreported for 1,2-diaminopropane ﬁlms irradiated with electrons (Wnuk etal.,2009).

5. Conclusions

We have undertaken a TEM investigation of hybrid (metal/ polymer)AuNPscappedwithPPAA,whichareofinterestfor appli-cationsinnanomedecine,suchasselectivetargetingoftumorsites. OurresultsshowthatthePPPAcoatingsofunstainedhybridAuNPs canbeimagedintheTEMwithsufﬁcientcontrastatan accelerat-ingvoltageof300kVatambienttemperaturewithouttheneedfor staining.ThethicknessofthePPAAcappingwastypicallybelow 2nmandoftennon-uniform.

FeaturesinEELspectraarelikelytobedominatedbythe sig-nalfromtheC supportﬁlm.Thisissuemightbealleviatedin a futurestudybyusinganimpregnationtechniqueforsupporting thecolloidofhybridnanoparticles(Gontardetal.,2014b).EFTEM imagesrecordedinthebulkplasmonenergyregimeshowedstrong intensityattheAu–polymerinterfaceandattheexternalsurface ofthepolymershell.Qualitatively,theresultspresentedhereshow thatlow-lossEFTEMcanbeanefﬁcientsourceofhighcontrastin imagesofhybridnanostructures.However,thefullinterpretation ofsuchimagesrequiresfurtherworkbecausetheplasmonmodes ofhybridcore–shellNPscannotbetreatedasadditiveindividual contributionsofthecoreandshellmodes(Chuntonovetal.,2012) andareverysensitivetosmallchangesinlocalmorphologyand electricalstate(Suetal.,2012;Jiangetal.,2014).

WhereasPPAAispredictedtobeelectron-beam-resistantatlow doses,thePPAAlayersofhybridNPsdamagedandbecame amor-phousunderelectronbeamirradiationat300keVwhenhighdoses wereused,althoughwecouldstillmeasurethepresenceofNin theshells.Knock-ondamageishighattheacceleratingvoltages usedinourwork.However,theuseofaCsupportﬁlmmayreduce sputteringfromtheshellsandexplaintheirstability.Ontheother hand,MonteCarlosimulationsshowthatthenumberofinelastic eventsclosetotheparticlesincreasesatlowaccelerationvoltages (≤20kV).Inafuturestudy,itwillbeofgreatinteresttocompare thepresentresultswithimagesacquiredatanintermediate accel-eratingvoltage(60–80kV).

Before the shells were destroyed, we observed the forma-tionofcrystallineorder aroundlargerNPs,aswellas graphitic planes.Polymericshellscontainingnitrogenwereobservedafter

irradiationwithveryhighelectrondoses,indicatingthatthe parti-clesarelikelytobemorestableattherelativelylowerdosesused inbiomedicalapplications.Also,suchexperimentsinvolvinginsitu TEMirradiationofpolymersmaybeimportantforunderstanding electron-beam-induced structural changes in polymers and for reﬁningapproaches fortuningtheopticalpropertiesofNPs.For example, polysterene homopolymer ﬁlms have been shown to becomeluminescentinvisiblelightunder50keVelectron irradi-ation,whichinducedthere-formationofaromaticrings(Leeetal., 2008).

Acknowledgment

We are grateful to the European Union for support under project reference REGPOT-CT-2011-285895-Al-NANOFUNC and underGrantAgreement312483–ESTEEM2(Integrated Infrastruc-tureInitiative–I3).

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