Removal of Protein Capping Enhances the Antibacterial Efficiency of Biosynthesized Silver

19 Pages·2015·2.21 MB·English

RESEARCHARTICLE
Removal of Protein Capping Enhances the
Antibacterial Efficiency of Biosynthesized
Silver Nanoparticles
NavinJain,ArpitBhargava,MohitRathi,R.VenkataramanaDilip,JitendraPanwar*
CentreforBiotechnology,DepartmentofBiologicalSciences,BirlaInstituteofTechnologyandScience,
Pilani,333031,India
* [email protected]
Abstract
Thepresentstudydemonstratesaneconomicalandenvironmentalaffableapproachfor
thesynthesisof“protein-capped”silvernanoparticlesinaqueoussolventsystem.Avariety
ofstandardtechniquesviz.UV-visiblespectroscopy,transmissionelectronmicroscopy
(TEM),energydispersivespectroscopy(EDS)andX-raydiffraction(XRD)measurements
wereemployedtocharacterizetheshape,sizeandcompositionofnanoparticles.The
synthesizednanoparticleswerefoundtobehomogenous,spherical,mono-dispersedand
coveredwithmulti-layeredproteinshell.Inordertopreparebaresilvernanoparticles,the
OPENACCESS
proteinshellwasremovedfrombiogenicnanoparticlesasconfirmedbyUV-visiblespec-
Citation:JainN,BhargavaA,RathiM,DilipRV,
troscopy,FTIRandphotoluminescenceanalysis.Subsequently,theantibacterialefficacyof
PanwarJ(2015)RemovalofProteinCapping
EnhancestheAntibacterialEfficiencyof protein-cappedandbaresilvernanoparticleswascomparedbybacterialgrowthrateand
BiosynthesizedSilverNanoparticles.PLoSONE10 minimuminhibitoryconcentrationassay.Theresultsrevealedthatbarenanoparticleswere
(7):e0134337.doi:10.1371/journal.pone.0134337
moreeffectiveascomparedtotheprotein-cappedsilvernanoparticleswithvaryingantibac-
Editor:AmitavaMukherjee,VITUniversity,INDIA terialpotentialagainstthetestedGrampositiveandnegativebacterialspecies.Mechanistic
Received:February21,2015 studiesbasedonROSgenerationandmembranedamagesuggestedthatprotein-capped
andbaresilvernanoparticlesdemonstratedistinctmodeofaction.Thesefindingswere
Accepted:July8,2015
strengthenedbytheTEMimagingalongwithsilverionreleasemeasurementsusinginduc-
Published:July30,2015
tivelycoupledplasmaatomicemissionspectroscopy(ICP-AES).Inconclusion,ourresults
Copyright:©2015Jainetal.Thisisanopenaccess illustratethatpresenceofproteinshellonsilvernanoparticlescandecreasetheirbacteri-
articledistributedunderthetermsoftheCreative
cidaleffects.Thesefindingsopennewavenuesforsurfacemodificationsofnanoparticles
CommonsAttributionLicense,whichpermits
unrestricteduse,distribution,andreproductioninany tomodulateandenhancetheirfunctionalproperties.
medium,providedtheoriginalauthorandsourceare
credited.
DataAvailabilityStatement:Allrelevantdataare
withinthepaperanditsSupportingInformationfiles.
Funding:Thisresearchworkwasfinancially Introduction
supportedbyUniversityGrantCommission(www.
ugc.ac.in),GovernmentofIndiaunderMajorProject Overthepastfewyears,worldwideescalationandaugmentationofmulti-drugresistancein
Scheme[F.No.42-185/2013(SR)]. microorganismshasbeenaseriousconcernformodernmedicine[1–5].Theneedforthe
developmentofnew,lowcostandeffectiveantimicrobialagentsindependentofbacterialresis-
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist. tancehasrevivedtheinterestofscientificcommunitytoexploretheantimicrobialpropertiesof
PLOSONE|DOI:10.1371/journal.pone.0134337 July30,2015 1/19
SurfaceModificationEnhancesAntibacterialActivityofAgNPs
silveranditscompounds.Inpastfewyears,varietyofnewsilverformulationssuchassilver
sulfadiazine,silvercitrate,silverlactate,etc.havebeendevelopedandvarioussilverintegrated
formulations(Katadyn,Argyrol,Movidyn,Tetrasil,Alagon,etc.)werecommercialized[6].
However,costconcernandrecentreportsofsilver-resistantbacterialstrainshaslimitedthe
useofsilverasapotentialalternativetoantibiotics[7].
Since,mostofthebiologicalprocessestakeplaceatthenanoscalelevel,acombinedapplica-
tionofbiologyandnanotechnologycanperhapsmeetthischallenge[8].Utilizationofsilver
nanoparticlescanbeparticularlyadvantageousascomparedtotheirbulkcounterpartasthe
formermanifesthighsurfaceareatovolumeratiowhichcanprovidebettercontactwithmicro-
organisms.Ithasbeendemonstratedthatsilvernanoparticlesrestrictthemicroorganismsto
developresistance[9–11].Additionally,atlowconcentrationssilvernanoparticleshavebeen
reportedtobenon-toxictohumancells,andhenceconsideredasansafeantimicrobialagent
[12].Owingtotheseproperties,anenormousincreaseintheapplicationsofsilvernanoparti-
clesforawiderangeofmedicalandcommercialproductshasbeenobservedwhichincludes
householdantisepticspraysandantimicrobialcoatingsformedicaldevicesthatsterilzeairand
surfaces[13].Silvernanoparticleshavealsoexpandedtheirhorizonsintextiles,cosmetics,air
purifiers,foodpackaging,coatingforrefrigerators,waterdisinfection;infact,ineveryapplica-
tionwherebacteriamayexertaharmfuleffect[14].
Themechanismofbactericidaleffectsofsilvernanoparticleshasnotbeenyetcompletely
elucidated.Inparticular,thereisadebateastowhetherthetoxicityisonlyduetothesilver
nanoparticlesoritisconfinedtothesilverionsreleasedfromthesenanoparticles.Ithasbeen
reportedthatthereleaseofsilverionsfromthecrystallinecoreofsilvernanoparticlescontrib-
utetothebactericidaleffects[15].Inaerobicconditions,silvernanoparticlesgetoxidised
releasinghighconcentrationsofsilverionsinsolution,whichinteractswithproteinsgiving
risetobactericidaleffects[16].Theantibacterialactivityofmicromolarconcentrationofsilver
ionsmaybelinkedwithuncouplingofrespiratoryelectrontransportfromoxidativephosphor-
ylation[17],inhibitionofrespiratorychainenzymesandinterferencewiththemembraneper-
meability[18]orinteractionwithcytoplasmiccomponentsandnucleicacids[19].However,
themolecularmechanismbehindthebactericidalactivityofsilverionsisstillnotclear.Inaddi-
tion,whetherthebactericidaleffectsareexertedbynanoscalephnemenonisanelusiveques-
tion.Xiuetal.[20]attemptedtodecipherthischallengebyperformingantibacterialstudies
understrictanaerobicconditionstoprecludesilveroxidationandAg+release.Theirfindings
completelyruledoutthedirectparticle-specificbactericidaleffectsofsilvernanoparticlesand
inferredAg+ionsasthedefinitivemoleculartoxicantforbactericidaleffects.Thus,astrictcon-
troloverthereleaseofsilverionsisapre-requisitefortheantibacterialefficacyofsilvernano-
particles.Manipulationatvariouslevelssuchasparticlesize,morphology,surfacecharge,
coatingandoxygenavailablityhavebeenconsideredasimportantparameterstocontroland
modulatetheantibacterialactivityofsilvernanoparticles.Amongtheseparameters,surface
coating(orfunctionalization)serveasthemostimportantfactorwhichdeterminethenano-
particle-microbeinteractions.Ithasbeenobservedthatcoatingofsilvernanoparticleswith
surfactantsresultsingreaterdamagetomicroorganismsincomparisontosimilar-sizedbare
silvernanoparticles[21,22].Similarly,comparitivestudiesofpolysaccharidecoatedandbare
silvernanoparticlesshowedthatpresenceofpolysaccharidemoleculesonnanoparticlesurface
facilitatesthedamagetomammaliancell-lines[23].Conversely,nanoparticleswerefoundto
belesstoxictomicroorganismswhencoatedwithpolymersornaturalorganicmatter[24].
Variegatedeffectsofsurfacechemistryonsilvernanoparticleuptakeandtoxicityhavealso
beenreported.Forinstance,presenceofmoleculessuchasalbumin,lecithin,polysorbital-80
andpeptideonthenanoparticlesurfacehavebeenreportedtofacilitatetheiruptakeandtoxic-
ity,whilepolyethyleneglycolinterferedwithuptakeinthelivercells[25].Incontrast,a
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SurfaceModificationEnhancesAntibacterialActivityofAgNPs
systematicsub-chronicreproductiontoxicityassessmentofsilvernanoparticlescoatedwith
polyvinylpyrrolidone(hydrophilic)oroleicacid(amphiphilic)onsoilearthworms(Eisenia
fetida)showednosignificantdifferencesinsilveraccumulationortoxicity[26].Hence,studies
targetedtounderstandthedynamicbehaviourofnanoparticlecoatings(inorganicororganic)
couldbehighlyinformativefordesigningefficientantibacterialformulationsofsilver
nanoparticles.
Inthepresentstudy,wearereportingaone-stepprotocoltosynthesize“protein-capped”
silvernanoparticlesusingAspergillussp.NJP02.Thepresenceofproteinshellonnanoparticles
ishighlyadvantageousasitimpartssolubilityandcolloidalstabilityinaqueoussystem.In
ordertofindouttheroleofproteinshellinmodulatingantibacterialefficacy,thecomparative
antibacterialpotentialofprotein-cappedandbaresilvernanoparticleswereassessed.Our
resultsclearlyindicatethatthepresenceofproteinshelloverthesurfaceofsilvernanoparticles
negativelyaffecttheirantibacterialpotential.Furthermechanisticstudiessuggestedthatpro-
tein-cappedandbaresilvernanoparticlesdemonstratedistinctmodeofaction.
Experimental
FungusAspergillussp.NJP02(GenBankaccessionnumber:HM222932)wasusedfortheextra-
cellularsynthesisofsilvernanoparticles.Thecompletedetailsforisolationandmolecularchar-
acterizationoftheisolatecanbeobtainedfromourpreviousreport[27].
Synthesisandcharacterizationofprotein-cappedandbaresilver
nanoparticles
Theextracellularsynthesisofsilvernanoparticleswasachievedusinganindigenousprotocol
developedinourlaboratory[28].Briefly,thefunguswasgrowninMGYPmediumfor72hat
28°Conarotaryshaker(150rpm)underdarkconditions.Afterincubation,thefungalmycelia
wereseparatedandwashedthricewithsterilewaterinordertoremovealltracesofmedia.Typ-
ically,10gofbiomass(freshweight)wasresuspendedin100mLofsteriledeionizedMilli-Q
waterandfurtherincubatedfor72hunderthesameconditionsasdescribedabove.Afterincu-
bation,biomasswasseparatedbyfiltrationusingWhatmanfilterpaperno.1andthefungal
cellfreefiltratecontainingextracellularsecretionswascollected.Forsynthesisofsilvernano-
particles,aqueoussilvernitratesolutionatafinalconcentrationof1.0mMwasaddedtothe
reactionvesselscontainingcell-freefiltrateandincubatedat28°Conarotaryshaker(150rpm)
withoutlight.Theobtainedprotein-cappedsilvernanoparticleswereusedforfurtherexperi-
ments.TheparticleswerecharacterizedbyUV-visiblespectroscopy,dynamiclightscattering
(DLS),transmissionelectronmicroscopy(TEM),energydispersivespectroscopy(EDS)and
selectedareaelectrondiffraction(SAED)analysis.Thedetailedproceduresfortheaboveanaly-
seshavebeendiscussedinourpreviousreport[28].Thecrystallinephaseofnanoparticleswas
measuredbyX-raydiffraction(XRD)studiesusingaRigakuMiniFlex-IIbenchtopdiffractom-
eteroperatedatavoltageof40kVandcurrentof30mAwithCuKαradiation.Inorderto
checkthestabilityofprotein-cappedsilvernanoparticles,UV-visiblespectrumofthreemonth
oldsamplewasalsorecorded.
Forpreparationofbaresilvernanoparticles,theas-synthesizedprotein-cappedsilvernano-
particlesolutionwascentrifugedat10,000rpmfor20min.Thepelletwassuspendedin1%
(w/v)sodiumdodecylsulphate(SDS)andboiledinwaterbathfor30mininordertodetach
theproteinshellfromnanoparticlesfollowedbycentrifugationat10,000rpmfor20min.The
supernatantcontainingtheunreactedSDSandSDS-proteincomplexwasanalyzedforthe
presenceofproteinsbymeasuringtheUV-visibleabsorptionspectrum.Theresultingpellet
wasboiledin1mLofTris-Cl(pH8.0)inwaterbathfor10mintoeliminatethepossibilityof
PLOSONE|DOI:10.1371/journal.pone.0134337 July30,2015 3/19
SurfaceModificationEnhancesAntibacterialActivityofAgNPs
SDSbindingtothenanoparticles,ifany.ToensurethecompleteremovalofSDS,dialysiswas
carriedoutagainstMilli-Qwaterwithfourchangesofwater.Theobtainedbaresilvernanopar-
ticleswerecharacterizedusingFouriertransforminfraredspectroscopy(FTIR),Photolumines-
cence(PL)spectroscopy,UV-visiblespectroscopyandDLSmeasurements.FTIRspectraof
freeze-driedsampleswererecordedonaShimazduIRPrestige-21FTIRspectrometer.Photolu-
minescencemeasurementswereperformedonaHoribaFluroMax-4spectrofluorometerwith
anexcitationwavelengthof280nmusing90°illumination.Inordertoachievemaximal
signal-to-noiseratio,excitationandemissionslitwidthvalueswereattunedto2.5and3.0nm,
respectively.UV-visiblespectroscopymeasurementswerecarriedoutonaJascoV-630UV-
visiblespectrophotometerataresolutionof1nm.DLSmeasurementswereconductedusinga
MalvernZetasizerNanoZSinstrumentandtheobtaineddatawereanalysedusingZetasizer
software.
Antibacterialefficiencyofsilvernanoparticles
Invitrobactericidaleffectsofprotein-cappedandbaresilvernanoparticleswereevaluated
againstclinically-importantbacterialpathogensprocuredfromtheInstituteofMicrobialTech-
nology,India.ThetestedstrainswereGrampositiveBacilluscereus(MTCC430)andPseudo-
monasputida(MTCC102);andGramnegativeEscherichiacoli(MTCC1652)andKlebsiella
pneumoniae(MTCC432).Theselectedbacterialspecieswereexposedtoprotein-cappedor
baresilvernanoparticles(50μgnanoparticlespermLofmedium)for30minat37°Cunless
otherwisestated.
Assaysforantibacterialactivity. Toexaminetheeffectonbacterialgrowthrate,the
selectedbacterialspeciesweregrownseparatelyin100mLofnutrientbrothsupplemented
with50μgofprotein-cappedorbaresilvernanoparticlespermLofmedium.Thebacterial
growthratesweredeterminedbymeasuringtheabsorbanceat600nmatdifferenttimeinter-
vals(0.1absorbancecorrespondstoaconcentrationof108cellspermL).Mediawithoutnano-
particlesandbacterialcellswereusedaspositiveandnegativecontrols,respectively.
Minimuminhibitoryconcentration(MIC)valuesofprotein-cappedandbaresilvernano-
particlesweredeterminedbyperformingdehydrogenaseassayina96-wellplate.Bacterial
inoculumswerepreparedbywashingtheovernightgrownculturetwicewithphosphatebuffer
saline(pH7.4)followedbydilutiontoachieveafinalconcentrationof108cfumL-1.100μLof
bacterialinoculumand20μLoftriphenyltetrazoliumchloride(TTC;3mgmL-1solution)
wereaddedineachwell.Subsequently,20μLof50μgmL-1silvernanoparticle(protein-capped
orbare)solutionwasaddedandtheplateswereincubatedfor18hoursat37°Cunderdarkcon-
ditions.Thetriphenylformazan(TPF)formedwasmeasuredat485nmusinganELISAreader.
TheMICassaywasdoneintriplicateandexecutedthricetovalidatetheMICvaluesforthe
eachtestedbacterialspecies.
Assaysforantibacterialmechanism. Thegenerationofintracellularreactiveoxygenspe-
cies(ROS)inpresenceofprotein-cappedorbaresilvernanoparticleswasdeterminedusingan
oxidation-sensitivefluorescentdye2,7-dichlorodihydrofluoresceindiacetate(DCFH-DA)in
triplicateasperthestandardprotocol[29].Briefly,theovernightgrownbacterialcells(108cfu
mL-1)werewashedthricewithphosphate-bufferedsaline(pH7.4)andfurthersuspendedin
freshnutrientbroth.DCFH-DA(10μMinphosphate-bufferedsaline)wasmixedinthe
mediumataratioof1:2,000(dye:medium)andincubatedfor30minat37°Cindarkcondi-
tionstosuccessfullypenetratethedyeintothebacterialcells.TheDCFH-DAloadedcellswere
separatedfromthefreedyemoleculesbycentrifugationat8,000rpmfor5minfollowedbya
finalwashwiththephosphate-bufferedsaline(pH7.4).Thebacterialpelletswereexposedto
protein-cappedorbaresilvernanoparticlesasmentionedearlier.Inlivingbacterialcells,
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SurfaceModificationEnhancesAntibacterialActivityofAgNPs
intracellularesterasesresultinhydrolysisofDCFH-DAtonon-fluorescent2,7-dichlorodihy-
drofluorescin(DCFH)whichinpresenceofROSoxidizestofluorescentdichlorodihydrofluor-
escin(DCF)[30].ThefluorescentsignalintensityofDCFwasmeasuredbyPerkinElmer
VICTORXMultilabelPlateReaderatanexcitationandemissionwavelengthof485nmand
535nm,respectively.Bacterialcellsincubatedin25mMascorbicacidfor1hbeforeDCFH-DA
exposurewereusedaspositivecontrolwhilecellswithoutDCFH-DAtreatmentwereusedas
negativecontrol[31].
Peroxidaseandsuperoxidedismutase(SOD)activitiesweremeasuredintriplicatefollowing
themethodofHochmanandGoldberg[32]andAroraetal.[33],respectively.Afterexposure,
thebacterialcellswerecentrifugedat8,000rpmfor5minandthepelletwassonicatedto
obtainthecrudeenzymeextract.Bacterialculturewithoutnanoparticletreatmentservedas
control.
Reactionmixturefortheassayofperoxidasecontained1mLof0.01Mpyrogallol,2mLof
0.1Mphosphatebuffer(pH6.0),and1mLof5mMhydrogenperoxide[32].Thereactionwas
initiatedbyadding1mLofenzymeextractandthemixturewasincubatedat25°Cfor5min.
Subsequently,thereactionwasterminatedbyadding1mLof2.5NH SO andtheamountof
2 4
purpurogallinformedwasestimatedbymeasuringtheabsorbanceat420nm.Inblank,1mL
ofsterilewaterwasusedinsteadofextract.Oneunitofperoxidasewasdefinedastheamount
ofenzymerequiredtoform1mgofpurpurogallinperminunderthespecifiedconditions.
TheSODassayisbasedontheabilityofSODtoinhibitthephotochemicalreductionof
nitrobluetetrazolium(NBT)[33].ReactionmixturefortheassayofSODcontained13.0mM
methionine,6.3μMNBT,6.5μMriboflavin,0.1mMEDTA,and50mMphosphatebuffer(pH
7.8).Thereactionwasinitiatedbyadding500μlenzymeextract(diluted10times)to1.5mLof
reactionmixturefollowedbyincubationat30°Cfor10minunder6000luxlightintensity.
Afterincubation,thetubeswereimmediatelytransferredtodarkconditionsandtheabsor-
bancewasmeasuredat560nm.Reactionmixturecontainingsterilewaterinsteadofenzyme
extractservedasblank.Thenon-irradiatedreactionmixtureservedasnegativecontrol.One
unitoftheSODactivitywasdefinedastheamountofenzymerequiredtoinhibitthereduction
ofNBTby50%underthespecifiedconditions.
Malondialdehyde(MDA)formation,ameasureofmembranedamagewasmonitoredusing
thiobarbituricacid(TBA)asaprobemolecule[34].Afterexposuretoprotein-cappedorbare
silvernanoparticles,thebacterialcellswerehydratedin1mLof2.5%(w/v)trichloroaceticacid
(TCA)andsubsequentlycentrifugedat12,000rpmfor20minat4°C.Thesupernatant(100μl)
wasmixedwith0.5%(w/v)TBAreagentpreparedin20%(w/v)TCA.Thereactionmixture
washeatedat100°Cfor30mininawaterbathfollowedbycentrifugationat12,000rpmfor
10minat4°C.TheabsorbanceoftheMDA-TBAadductwasmeasuredat532nm.MDAcon-
tentwasexpressedaspicomolespermgofproteinusingamolarextinctioncoefficientof1.56
mM-1cm-1[34].Theextentofmembraneleakagewasalsodeterminedbyquantificationof
protein[35],totalsugar[36]andnucleicacidcontent.Thenucleicacidleakagewasdetectedby
measuringtheabsorbanceofculturesupernatantat260nm(1ODunit=50μgmL-1nucleic
acid).
Inductivelycoupledplasmaatomicemissionspectroscopy(ICP-AES)measurements.
Thedissolutionofsilverionsfromprotein-cappedorbaresilvernanoparticleswasmeasured
innutrientmediumafterexposuretobacterialcells.Thesilverionswereseparatedbydialyzing
theculturemediumagainststerileMilli-Qwaterfor12husinga12kDacellulosemembrane
andmeasuredbyShimadzuICPS-7500instrument.Thesilverionconcentrationinnutrient
medium(withoutsilvernanoparticles)wasusedascontrol.
TEMimaging. InordertopreparesampleforTEMimaging,nanoparticletreatedbacterial
cellswerecentrifugedat5,000rpmfor2min.Thebacterialpelletswerewashedtwicewith
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SurfaceModificationEnhancesAntibacterialActivityofAgNPs
Fig1.UVvisiblespectrumofreactionmediumasafunctionoftime(0,12,24,48and72h).Insetshows
tubescontainingfungalcell-freefiltrate(a)withoutand(b)withsilvernitratesolutionafter72hofreaction.
doi:10.1371/journal.pone.0134337.g001
phosphatebuffersaline(pH7.2)followedbyapre-fixationstepwith2.5%glutaraldehyde(pre-
paredin0.1Mcacodylatebuffer)for2hat4°C.Post-fixationofthebacterialcellswasper-
formedwith1%OsO treatmentfor1hat4°C.Afterpost-fixation,theOsO solutionwas
4 4
removedbywashingtwicethebacterialcellswith0.1Mcacodylatebufferfollowedbystaining
withfiltereduranylacetate(1%)solution.ForTEMimaging,adropofstainedbacterialsample
wasplacedonacarbon-coatedcoppergridanddriedovernightinavacuumdesiccator.Thegrids
wereobservedonaHitachiH-7650TEMinstrumentoperatedataconstantvoltageof100kV.
ResultsandDiscussion
Characterizationofprotein-cappedandbaresilvernanoparticles
Agradualchangeinthecolourofreactionmedium(containingfungalcell-freefiltrateand
precursorsilverions)fromcolourlesstoreddishbrownrevealedavisualevidenceforsilver
nanoparticlesynthesis(Fig1inset).UV-visiblespectrumshowedagradualincreaseinthe
absorbanceat429nmwithrespecttotime(Fig1).Theabsorptionmaximaat429nmcanbe
attributedtothesurfaceplasmonresonance(SPR)vibrationsofsynthesizedsilvernanoparti-
cles[37].Nofurtherincreaseinabsorbancewasobservedafter72hofreaction(datanot
shown),whichindicatedthecompletereductionofprecursorsilverionsinreactionmedium.
Stabilityofas-synthesizedsilvernanoparticleswasmonitoredperiodicallyformorethanthree
months.Itwasobservedthatthenanoparticlesolutionwasextremelystableatroomtempera-
ture,withnoevidenceofparticleaggregationasdeterminedbyUV-visiblespectroscopy
measurements.
TEMmicrograph(Fig2A)revealedthepresenceofmono-dispersedandpredominantly
sphericalparticleswithnovisibleaggregation.TheSAEDpattern(Fig2Ainset)atteststhe
crystallanityofsilvernanoparticles.Theparticlesizedistributionhistogramobtainedfrom
TEMmeasurementsrevealedthatmostoftheparticlesrangedbetween40–80nmwithamean
diameterof54±8.9nm(Fig2B).Theseresultswereinwellagreementwiththevaluesobtained
byDLSmeasurements(Fig2C).Ithasbeenwellreportedthatdifferentfungicansynthesize
nanoparticlesofvariedcomposition,sizesandshapeswhichmaybeduetothedifferencesin
theirextracellularproteinprofiles[38–40].
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SurfaceModificationEnhancesAntibacterialActivityofAgNPs
Fig2.(A)Arepresentativetransmissionelectronmicrographshowingsphericalshapedsilver
nanoparticles(scalebarequivalentto50nm).InsetshowingSAEDpatternrecordedfromasingle
nanoparticle.Particlesizedistributionhistogramofsilvernanoparticlesasdeterminedusing(B)
transmissionelectronmicroscopeand(C)dynamiclightscatteringmeasurements.
doi:10.1371/journal.pone.0134337.g002
X-raydiffractionpatternrecordedbypreparingdrop-coatedfilmofprotein-cappedsilver
nanoparticlesfurthervalidatedthecrystallinenatureofnanoparticles.Thewell-definedpeaks
at2θvaluesof38.03°,46.18°,64.60°,and77.18°correspondsto(111),(200),(220)and(311)
planesofsilver,respectively(Fig3A).Thesevalueswereincompleteagreementwiththeface-
centeredcubic(fcc)latticestructureofcrystallinesilver(JCPDSfileno.04–0783).Asimilar
patternofXRDspectrumhasbeenreportedformetallicsilvernanoparticlessynthesizedby
otherfungi[41].EDSwascarriedouttodeterminetheelementalcompositionofas-synthe-
sizednanoparticlesandcappingmolecules.Anintenseopticalabsorptionbandat3.0KeV
Fig3.(A)XRDspectrumofas-synthesizedprotein-cappedsilvernanoparticleswithBragg’s
diffractionvaluesshowninparentheses.(B)EDSspectrumshowingtheelementalcompositionof
silvernanoparticles.
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SurfaceModificationEnhancesAntibacterialActivityofAgNPs
Fig4.(A)FTIRspectraand(B)Photoluminescencespectraofprotein-cappedandbaresilver
nanoparticles.
doi:10.1371/journal.pone.0134337.g004
confirmedthepresenceofpuremetallicsilvernanoparticles(Fig3B).Otherpeaksobservedfor
C,NandOatomsindicatedthepresenceofproteinsascappingmolecules.
UV-visibleabsorptionspectrumofsupernatantobtainedafterSDStreatmentofassynthe-
sizedsilvernanoparticlesshowedabroadabsorptionpeaknear280nmwhichcorrespondsto
aromaticaminoacidsofproteinsS1Fig[28].FTIRandphotoluminescencemeasurementsof
protein-cappedandbaresilvernanoparticleswerecarriedouttoconfirmtheremovalofcap-
pingproteinsfromthesurfaceofsilvernanoparticles.FTIRspectrumofprotein-cappedsilver
nanoparticles(Fig4A)exhibitedcharacteristicbandsofamideIandamideIIatwavenumbers
1651and1539cm-1,respectivelyandC-Nstretchingvibrationbandofaliphaticamineat1029
cm-1[28].ThedisappearanceofthesecharacteristicproteinbandsintheFTIRspectrumof
baresilvernanoparticlesclearlyindicatedtheremovalofproteinsfromnanoparticles.ThePL
spectrum(Fig4B)ofprotein-cappedsilvernanoparticlesshowedadistinctemissionpeakat
340nm,whichcouldbeattributedtothetyrosineresiduesofcappingproteins.Incontrast,
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SurfaceModificationEnhancesAntibacterialActivityofAgNPs
Fig5.(A)UVvisiblespectraand(B)particlesizedistributionofprotein-cappedandbaresilver
nanoparticles.
doi:10.1371/journal.pone.0134337.g005
absenceofemissionpeakincaseofbaresilvernanoparticlesconfirmedthecompleteremoval
ofproteinmolecules.
TheremovalofproteinshellbySDStreatmentreducedthesizeofnanoparticlesasreflected
bytheblueshiftobservedintheSPRpeakfrom429to425nmincaseofbaresilvernanoparti-
cle(Fig5A).Calzolaietal.[42]reportedasimilarobservationwhilestudyinginteraction
betweenhumanubiquitinandgoldnanoparticles.Furthermore,hydrodynamicparticlesize
distributionandpolydispersityindex(PDI)analysiswascarriedouttoinvestigatetheoccu-
pancyofproteinshellincaseofprotein-cappedsilvernanoparticles.Theprotein-cappedsilver
nanoparticlesshowedameanparticlesizeof90.53nm(PDI=0.357)whichdecreasedto58.39
nm(PDI=0.396)incaseofbaresilvernanoparticlesindicatingthesuccessfulremovalofpro-
teinshellfromnanoparticlesafterSDStreatment(Fig5B).Theplausiblereasonformulti-lay-
eredshell(~32nmthick)couldbethenon-specificandnon-competitivebindingofproteins
presentinthesurroundingenvironment(reactionmedium).Thepersistenceofthickprotein
shellonbiogenicsilvernanoparticleshasattractedourattentiontocomparetheantibacterial
efficacyofprotein-cappednanoparticlesincomparisontobaresilvernanoparticles.
Antibacterialefficacyofprotein-cappedandbaresilvernanoparticles
Thetime-dependentbacterialgrowthinpresenceofprotein-cappedandbaresilvernanoparti-
cleswasmonitoredbymeasuringtheabsorbanceat600nm[43].Theprotein-cappedaswellas
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