DTIC ADA605469: Hybrid Antimicrobial Enzyme and Silver Nanoparticle Coatings for Medical Instruments (Postprint)

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by  Defense Technical Information Center

AFRL-RX-TY-TP-2008-4630

HYBRID ANTIMICROBIAL ENZYME AND SILVER
NANOPARTICLE COATINGS FOR MEDICAL
INSTRUMENTS (POSTPRINT)

D. Mathew Eby and Heather R. Luckarift
Universal Technology Corporation
1270 North Fairfield Road
Dayton, OH 45432

Glenn R. Johnson
Airbase Technologies Division
Air Force Research Laboratory
139 Barnes Drive, Suite 2
Tyndall Air Force Base, FL 32403-5323

Contract No. FA4819-07-D-0001

December 2008

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4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER
Hybrid Antimicrobial Enzyme and Silver Nanoparticle Coatings for Medical FA4819-07-D-0001
Instruments (POSTPRINT)
5b. GRANT NUMBER
5c. PROGRAM ELEMENT NUMBER
0909999F
6. AUTHOR(S) 5d. PROJECT NUMBER
*Eby, D. Matthew; *Luckarift, Heather R.; ^Johnson, Glenn R. 4918
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L0
5f. WORK UNIT NUMBER
Q140LA62
7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION
REPORT NUMBER
*Universal Technology Corporation
1270 North Fairfield Road
Dayton, OH 45432
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Document contains color images. Ref Public Affairs Case # AFRL/RXQ 08-203.
Published on http://pubs.acs.org | doi: 10.1021/am9002155.
14. ABSTRACT
We report a method for the synthesis of antimicrobial coatings on medical devices that combine the bacteriolytic activity of
lysozyme and the biocidal properties of silver nanoparticles. Colloidal suspensions of lysozyme and silver nanoparticles were
electrophoretically deposited onto the surface of stainless steel surgical blades and needles. The coatings retained the hydrolytic
properties of lysozyme and exhibited antimicrobial activity against a range of bacterial species. In particular, coated blades
demonstrated potent bactericidal activity, reducing cell viability by > log 10 within 1.5 h for Klebsiella pneumonia, Bacillus
anthracis Sterne and Bacillus subtilis and within 3 h for Staphylococcus aureus. The results confirmed that complex antimicrobial
coatings can be easily made using facile silver nanoparticle synthesis and electrodeposition methods.
15. SUBJECT TERMS

antimicrobial, lysozyme, silver, nanocomposite, electrochemistry
16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF 18. NUMBER 19a. NAME OF RESPONSIBLE PERSON
a. REPORT b. ABSTRACT c. THIS PAGE ABSTRACT OF Glenn R. Johnson
PAGES
UU 19b. TELEPHONE NUMBER (Include area code)
U U U 8
Standard Form 298 (Rev. 8/98)
Reset
Prescribed by ANSI Std. Z39.18
Hybrid Antimicrobial Enzyme and Silver
Nanoparticle Coatings for Medical
A
R
Instruments
T
I
C
D. Matthew Eby,*,†,‡ Heather R. Luckarift,†,‡ and Glenn R. Johnson*,‡ L
E
MicrobiologyandAppliedBiochemistry,MaterialsandManufacturingDirectorate,AirForceResearchLaboratory
andUniversalTechnologyCorporation,139BarnesDrive,Suite2,TyndallAFB,Florida32403
ABSTRACT Wereportamethodforthesynthesisofantimicrobialcoatingsonmedicalinstrumentsthatcombinesthebacteriolytic
activityoflysozymeandthebiocidalpropertiesofsilvernanoparticles.Colloidalsuspensionsoflysozymeandsilvernanoparticles
wereelectrophoreticallydepositedontothesurfaceofstainlesssteelsurgicalbladesandneedles.Electrodepositedfilmsfirmlyadhered
tostainlesssteelsurfacesevenafterextensivewashingandretainedthehydrolyticpropertiesoflysozyme.Theantimicrobialefficacy
ofcoatingswastestedbyusingbladesandneedlesinaninvitrolyticassaydesignedtomimicthenormalapplicationoftheinstruments.
Coatedbladesandneedleswereusedtomakeincisionsandpunctures,respectively,intoagaroseinfusedwithbacterialcells.Cell
lysiswasseenatthecontactsites,demonstratingthatantimicrobialactivityistransferredintothemedia,aswellasretainedonthe
55 surfaceofthebladesandneedles.Bladecoatingsalsoexhibitedantimicrobialactivityagainstarangeofbacterialspecies.Inparticular,
1
1, 2009am9002 tcBhoaaactitlecludosmbalpandltehexrsaadcniestmiSmoteincrsrntoerba,itaaenlddcpoBoaattceininlgltusbscasacuntbetbriilecisicdaraneladatcwetdiivthuitsiyni,n3rgehfdafucocirliSentgmapceehtlyhllvooidcaosbcifcloiutryssabiulyvreaerutsnleaaansnotdp3Aalorctigincweletiotsbhyainnctt1her.e5bsihasyfaloynrid.KTlehelbeescriteerlsoluadletpspncoeousnimtfiioornnmi.aeed,
y 321/
AB on Juldoi: 10.10 KEYWORDS: antimicrobial•lysozyme•silver•nanoparticle•electrochemistry•coating
CH Lorg | INTRODUCTION the antimicrobial effectiveness of silver coatings has been
ARacs. Infections originating from implanted devices (e.g., or- showninthelaboratory,therehavebeenmixedresultsin
SEbs. thopedicfixationhardware,artificialprosthetics,endo- theclinicalsetting(7).Thepastlimitationsofsilvercoatings
E REp://pu vascular stents, and catheters) are a persistent and asprophylactictreatmentsmostlikelystemfromtheinef-
RChtt serioushealthissue.Thefirststeptowardsreducingthese fective release of silver ions from the surface and the
AIR FO2009 on ionufsecdteiovnicseissuirnfhaicbeisti.nCgubrraecntetrainalticmoliocrnoizbaiatilocnoaotninsgumbceutthaondes- ubnodpyroflduuicdtsiv(e8,a9d)s.orptionandprecipitationofsilverionsin
ed by uly 2, ionfvtohlevedeeivthiceermsuordfaificecaotiroanpopfltichaetpiohnysoifocchoeamtinicgasltphraotperertsiiesst shoMwonretoreexcheinbtiltye,nshialvnecredinanntaimnoicpraorbtiiaclleefffoecrmtsohvaesrpbreeevni-
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oh
Dblis biocideandalsoprovidesustainedantimicrobialactivity,so withthebacterialcellthatisnoteasilyquenchedbyother
Pu toneutralizeanyinitialcontaminationofthesurfaceandalso salts and proteins (10, 11). Numerous methods for silver
inhibit colonization over time from a wide range of op- nanoparticlesynthesisandincorporationintomaterialsare
portunistic microorganisms. These issues and the rise of currentlyunderinvestigation(12-20),andsilvernanopar-
microbialresistancetocommonlyusedantibioticsdrivea ticle coatings are beginning to see more prevalent use in
continuoussearchfornewantibioticformulationsthatare healthcare(1,5,8).Inrecentwork,wediscoveredthatthe
amenabletoadhesionandimmobilizationonsurfaces. hydrolytic enzyme, lysozyme, catalyzes the reduction of
Onestrategygivenconsiderableattentioninrecentyears silver, which yields a stable colloid of silver nanoparticles
istheuseofsilveronmedicaldevicesurfacesandinwound and active lysozyme (21). The use of lysozyme in the
dressings (2-4). These coatings are particularly attractive synthesisofsilvernanoparticlesisadvantageousbecauseit
becausesilverionsactagainstabroadrangeofbacteriaand combinestwodifferentbiocidalmechanismsintoonema-
yetarerelativelynontoxictomammaliancells(5,6).While terial;lysozymeprimarilyinhibitsgrowthoftheGram-type
positivestrainsthroughitsmuramidaseactivity(22),while
silvernanoparticlesinhibitgrowthofbothGram-typenega-
*Corresponding author. Tel.: [phone]. Fax: [phone]. Email:
[email protected](D.M.E.),[email protected](G.R.J.). tive and positive strains by inhibiting membrane function
ReceivedforreviewMarch30,2009andacceptedJune16,2009 and enzyme activity (23). In a few cases, the synthesis of
†UniversalTechnologyCorporation. bioactivecoatingshasbeenachievedbyaddingnoblemetal
‡MicrobiologyandAppliedBiochemistry,MaterialsandManufacturingDirector-
nanoparticlestoenzymesolutionsandelectrodepositingthe
ate,AirForceResearchLaboratory.
DOI:10.1021/am9002155 mixture onto surfaces (24-27). Because of the unique
©2009AmericanChemicalSociety propertiesofsilvernanoparticles,theirutilityastheprecur-
www.acsami.org VOL.1•NO.7• 1553–1560•2009 1553
PublishedonWeb07/02/2009
sorforelectrodepositedcoatingsmayproducemoreeffec- into 5 mL of the lysozyme-silver nanoparticle deposition
tiveantimicrobialactivitiesthanpreviousmethodsbasedon solution.Aplatinumwirewasalsosubmergedapproximately
1cmintothesolutionandservedasthesecondaryelectrode.
E conventional electrodeposition solutions (e.g., silver salt
Thedistancebetweeneachelectrodewasapproximately1cm.
L solutions).Herein,wereportamethodtoproduceantimi- A power source [1735 direct-current (dc) power supply, BK
C crobialcoatingsthroughelectrophoreticdepositionofsilver Precision,Placentia,CA]appliedadcvoltage(3V)for10min.
I nanoparticles formed from the lysozyme-mediated reduc- Afterelectrodeposition,bladesandneedleswererinsedexten-
T tionofsilverions.Theantimicrobialeffectivenessofcoated sivelywithultrapurewaterandstoredatroomtemperature.
R surfaceswastestedagainstavarietyofmicroorganismsto Threeadditionalelectrophoreticcoatingexperimentswere
A demonstratetheretentionofantimicrobialpropertieswithin completedtodeterminetheindividualeffectsthatlysozymeand
silvernanoparticleshaveondepositionandwhenlysozymeand
coatings.
silver acetate were combined in the deposition solution but
without prior formation of nanoparticles. For the first two
EXPERIMENTAL SECTION controlexperiments,lysozymeandsilvernanoparticleswere
Reagents and the Synthesis of Silver Nanoparticles. Hen usedinseparateelectrodepositionsolutions.Forelectrophoretic
eggwhitelysozyme(∼95%,∼50000unitspermgofprotein) deposition containing only lysozyme, the procedure was fol-
and silver(I) acetate (99.99%) were obtained from Sigma- lowedasdescribedabove,exceptasolutioncontaining5mg
Aldrich (St. Louis, MO). Unless stated otherwise, all other mL-1 lysozyme in 50% methanol served as the deposition
chemicalswereobtainedfromeitherSigma-AldrichorFisher solution.Forelectrophoreticdepositionprimarilycomposedof
Scientific(Pittsburgh,PA)andwereofthehighestpurityavail- silver nanoparticles, a dialysis method was used to remove
able.Forexperimentsmeasuringtheelectrochemicalproperties soluble lysozyme from the deposition solution that was not
oflysozyme,alysozymesolutionmadefromastockpowder tightlyassociatedwithnanoparticles(21).Priortodeposition,
wasdialyzedagainsttwochangesofultrapurewater(1:100,v/v) a5mLsolutionoflysozyme-silvernanoparticleswasplaced
155 usinga5000Damolecularweightcut-offdialysismembrane intoa25000Damolecularweightcut-offdialysisbag(Spectra/
AB on July 31, 2009doi: 10.1021/am9002 (tsslwAySoittsoipnistoorhe,izelncTvyudt,eXrmlutra)ran/eaoaPtd-pcvoieue-sartirrllcay2ev2etz0eew4lelrudahsn°ltooalCetyslnourseotoairinepeznosamyndtr3metotr0iihcnve,leeSesm5npors0eelgodu%cslituitlimdurtouitumoneLmansd-els1t(Mbwh2fuo0aaeiftrndlfhmieeqoicmrllugeaaoc(lem1nttItrnsdhLom.da-psun1TaMh)soloottw)lrsri(eweega1trsenia:ec,1nsdHm,dettveorhha/apuevedtosn)nee.-- vPwitt(n1hi/ocav0ilrart)edemcsdsreceegbfloorlpuumvnrotleto7sLrdsis2o-tteiil1ahlo)leesnatxcsonttopsherdonereelrt)smuaiiametiliovnnloeeevedncnredtdtratt,hoishcafmdaeerlyteetmaazspcltehlooaedlnjasyo(imt1atramiigomtioanyinuxieMonnedsfst)dostlmiylnsfuosoooto5fuilszou0rltyyntl%cyimsohoasnmeaznis.lnyvdeTogmethefhwreislasnye.norsarFefoeonosl1zouru(yp1Llsmtta:eheo1rddeef,-
CH Lorg | aodfd5edmtgotmheL-ly1solyzsyomzyemsoeluatniodn0to.5prmodMucseilavefirnaalcceotantceenintra5t0io%n immediatelyintheelectrophoreticcellintheabsenceoflight,
Rs. inordertopreventsilverreductionandsubsequentnanoparticle
E RESEAp://pubs.ac mpTinrheotethxhreiemasndauaotlrlte.kilnyTag2htl4ey4sh°osCotzoylutmaotliloeion-nwhsiwtibhlviaetespfrorctrehomcelilnoaptiiditeoaaxntliposoounfssseupidlnevtneitlsroiunosalnieng.whotpasafrosttrioclraeepsd-. fsoprePmchtayrtosiisoccnao.lpaynwdaSspceocmtrpolsecteodpiucsiCnhgaaraCcateryri3zaEtisopne.ctUroVp-hvoitsoibmle-
ORCn htt ElectrochemicalMeasurements.Electrochemicalmeasure- eAtteorm(VicafroiarcneImnci.c,roPsacloopAylt(oA,FCMA))imanadge0s.5wemreLoqbutaaritnzedcuuvseitntgesa.
AIR F2009 o mceellnattsrwoeormepteemrfopremraetdurienuasoinnge-aco7m57paVrAtmCeonmt,pthurtreaec-eeleScytsrtoedme NmaicnroossccooppeeVa,nedquaipPpiceodFworicthe astaMguelt(iVmeoedceoVInssctraunmnienngtsprIonbc.e,
ed by uly 2, pinogt-ednistikosetlaetct(rMoedtero(2h.m0mAGm,Hdiesrkisdaiua,mSewteitrz)ewrlaasndem).Aplosiylveedrarsottahte- Wforoo1d0bmuriyn,bNeYf)o.rBelaimdeasgwinegreinfirtsatpdpriinegdmunoddeer,vuascinuugmanate5tc0h°eCd
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DoPublish esulercetmroednet..BTehfeorweoerakcihngmeelaescutrroemdeenwta,sthsetawtioornkainrygadnudrianugxmiliaeray- w<1a0sinnmth.eTr2a0n0smkHiszsiroanngeel,ecatnrdonthmeircardoisucsoopfyth(TeEpMro)bweatsipcowmas-
pletedusinga100CXIIelectronmicroscopefromJEOLLtd.
electrodeswerecleanedbypolishingwith8000gritsandpaper
(Tokyo,Japan).AttenuatedtotalreflectanceFouriertransform
andthenrinsedwithultrapurewater.Noadditionalelectrolyte
was added to the potentiostat cell. Current-potential curves infrared (ATR FT-IR) spectroscopy was performed using a
NicoletFT-IR6700spectrophotometerequippedwithaSmart
(cyclicvoltammograms)weregeneratedbylinearvoltagesweeps
between-1and1Vat0.1Vs-1(vsAg/AgCl). Miraclesingle-bouncediamondATRaccessory(ThermoFisher
Electrophoretic Deposition of Lysozyme-Silver Nano- Scientific, Waltham, MA). The data collection was completed
particles. Surfaces to accept electrophoretic deposition were using OMNIC 2.1 software. Before measurement, coated and
stainlesssteelsurgicalblades(#10,ExelintInternationalCo.,Los uncoatedbladeswerewashedwithmethanolanddriedat50
Angeles,CA)andsyringeneedles(single-use,22Gauge,1.5in. °Cfor30min.ForATRFT-IRofthelysozymestockpowder,
length,BectonDickinsonCo.,FranklinLakes,NJ).Beforedepo- 10µLofalysozymepowderslurryinmethanolwasdriedon
sition,metalsurfaceswerecleanedandetched,usingaproce- theinstrumentcrystalbeforemeasurement.
dure similar to that described previously (28). Blades and Measurement of the Lysozyme Activity. Qualitative mea-
needles were cleaned by ultrasonication for 15 min each in surementofthelysozymeactivityincoatingswasadaptedfrom
dichloromethane,acetone,andwater.Cleansurfaceswerethen theMicrococcuslysodeikticuscelllysisassayinsolidmedia,first
etchedfor1mininasolutioncontaining0.25%hydrochloric describedbyFleming(29).Bladesandneedleswerefirstused
acidand2.5%nitricacid.Afteretching,metalsurfacepassiva- tocutandpuncture,respectively,solidmediaplatescontaining
tionwascompletedin40%nitricacidfor40min.Bladesand a suspension of 0.5 mg mL-1 M. lysodeikticus cells (Sigma-
needles were then extensively washed with ultrapure water, Aldrich)ina50mMpotassiumphosphatebuffer(pH8)and1%
dried,andstoreduntiluse. agarosebeforebeingplacedontopoftheplatesurface.Plates
A two-electrode electrophoretic cell was constructed that wereincubatedat37°Cfor16handperiodicallyinspectedfor
containedeitherabladeoraneedleastheworkingelectrode. celllysis,whichresultedinaclearingofthesolidagaratthe
Approximately1cmofthebladeorneedletipwassubmerged bladeandneedlecontactpoints.
1554 VOL.1•NO.7• 1553–1560•2009 Ebyetal. www.acsami.org
AntimicrobialActivityAssays.Thefollowingstrainsusedin
theantimicrobialassayswereobtainedfromtheAmericanType
CultureCollection(ATCC,Manassas,VA):Escherichiacoli(ATCC
A
25922),Staphylococcusaureus(ATCC25923),Klebsiellapneu-
moniae(ATCC4352),Pseudomonasfluorescens(ATCC13525), R
Candidaalbicans(ATCC10231),andStaphylococcusepidermidis T
(ATCC 14990). Bacillus anthracis Sterne strain 34F2 was ob-
I
tainedfromColoradoSerumCo.,Denver,CO(30).Acinetobacter C
baylyi strain ADP1 (31) was a gift from Dr. Ellen Neidle
L
(University of Georgia, Athens, GA). Luria-Bertani and Yeast
Mold (Difco Laboratories, Sparks, MD) were used to grow E
bacterialstrainsandC.albicans,respectively,inliquidandon
solidmediaplates(platesalsocontained1.5%agarose).
Atthestartoftheantimicrobialassay,coatedbladeswere
incubatedingrowthmediacontainingapproximately105cells
in the exponential growth phase (1 mL). Viable cells were
quantitatedafter0,1.5,and3hofincubationbyremoving100
µL from the cultures, spreading all or a serial dilution of the
aliquotontosolidmedia,andenumeratingcolony-formingunits
(cfu). After cultures were incubated for 24 h, blades were
removedfromculturesandplacedintofresh,sterilemedia(1
mL).Inaddition,theremaininggrowthmediafromtheblade
culturewasdilutedintofresh,sterilemedia(1:10dilution).The
5 two subcultures were incubated overnight and then visually
5
1, 2009am90021 irsneesppplaiecrcaattteeedsc.ufoUltrungrceroosawtteotdhe,.nResetucsrhueelttdshaabtrleacedtlhelssegawrveeewrreaignaeltshoofeaiantbclseueabnsactteethdorfeinae FIGURE1.Lysozyme-catalyzedsilvernanoparticles(A).TEMimage
y 321/ coatingmaterial,andtheetchedstainlesssteeldidnotadversely of lysozyme-silver nanoparticles (B). UV-visible spectrum of
CH LAB on Julorg | doi: 10.10 aRffEeSScytUntLhteThgSersoAiwsNthDa.nDdISCEUleScStIrOocNhemical Behavior of vnlsyoiosdrotbuzaapynlmrcsoeepd-meucscaiileexvisemcr(u3unr3mar)en.anoCtpto4anp2rts0eiicadnlkeemssri(snuChgn)o.dtwheianrtgosaiulvrpelcarsoamncedontiatitroeensaoslno(andneacsdehoaeebds-
ARacs. Lysozyme-SilverNanoparticles.Inourrecentwork, gray line in Figure 2B), the shift in the peak potential of
Es.
E RESp://pub wfroemfouthneditnhcautbsatatibolneocoflmloiicdrsoogfrasmilvserpenranmoipllailritteicrleqsufaonrtmitieeds lwysitohzylymsoezayfmteersailnvderfaodrmditsioannseuwggeesletscttrhoacthseilmveircaalslysoacciatitvees
RChtt oflysozymeandsilveracetatein100%methanol(21).The species. As seen with lysozyme alone, additional voltage
AIR FO2009 on gtorolwigthhtoafnsidlveornnlyanroepqaurirtiecdleslywsoaszydmepee,nwdehnicthupaocnteedxpaossuthree ciryreclveesrsailbsloysdheopwoseidtetdhaantdthfeorlmyseodzyamcoea-tisnilgveornctohmeeplleecxtrwodaes
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oh
Dblis oussolutioncontaining50%methanol(Figure1).Thefinal forvoltammericanalysisandtheelectrodeswerepolished
u
P solutioncontainedasufficientconcentrationofionicspecies betweeneachuse.Ineachcase,therewasnochangeinthe
to facilitate electrophoretic deposition without additional potentialandintensityofthecurrentpeaks,whichindicated
electrolytes,whilealsomaintainingastablesuspensionof that the electrochemical properties of lysozyme were not
nanoparticles. When stored in the dark at ambient condi- alteredduringandafternanoparticlesynthesis(resultsnot
tions,thelysozyme-silvernanoparticlescanbemaintained shown). When silver nanoparticles were present in the
asacolloidforseveralmonths. potentiostatcell,aresilientredfilmformedontheelectrode
The electrochemical properties of lysozyme were mea- surface during repeated voltage cycling, which required
suredusingcyclicvoltammetrywithasilverdiskelectrode. removal by physical abrasion using fine-grit sandpaper.
Lysozyme was oxidized at 745 mV and underwent two Takentogether,theseobservationsshowedthatlysozyme-
reductions at -640 and -800 mV (Figure 2A). Repeated silvernanoparticlesformacomplexthatwaselectrophoreti-
potentiostat cycling resulted in successively smaller peak cally deposited onto the electrode surface. The results led
currents,whichwereindicativeofirreversibledepositionof us to investigate whether the antimicrobial properties of
protein on the electrode surface (32). When silver acetate lysozymeandsilverwereretainedwithinthecoatings.
was added to lysozyme, voltammograms completed im- Electrophoretic Deposition of Lysozyme-
mediatelyaftermixingshowashiftinthereductionpeaks SilverNanoparticlesontoStainlessSteel.Stainless
oflysozymetowardalowercathodicpotential(Figure2B). steelsurgicalbladesandsyringeneedleswereinvestigated
Similarvoltammericbehaviorhasbeenpreviouslyreported asmodelsurfacesforthecoating.Anelectrodepositioncell
whenprotein-metalcomplexesformintheelectrochemical was constructed using a blade or needle as the working
cellandchangetheelectrochemicalpropertiesoftheindi- electrode,andthetipwassubmergedinthelysozyme-silver
www.acsami.org VOL.1•NO.7• 1553–1560•2009 1555
the protein secondary and tertiary structures will alter
hydrogen bonding between the CO and NH groups in the
E peptidebackbone,whichresultsinaberrationsoftheamine
L I, II, and III bands between 1200 and 1700 cm-1 (34). No
C such significant shifts were observed in this IR region
I betweenlysozyme-coatedbladesandthestockpowder.In
T addition, coated blade surfaces were analyzed at the na-
R nometer scale using AFM (Figure 3). A layer of spherical
A nanoparticles on the surface of coated blades was clearly
seeninimages.TheAFMimagesofetched,uncoatedblades
didnothavethesefeatures(resultsnotshown).Thenano-
particlesvisualizedinthecoatingweresimilarinshapeand
diameter to the silver nanoparticles observed in the TEM
imagesofthedepositionsolution(Figure1).Thenanopar-
ticleswerealsoreadilyseeninphaseimages(lighterareas,
Figure3E)andweresurroundedbyamaterialofcontrasting
phase(darkerareas).Undercloserinspection,edgeenhance-
ment illustrated in the 200 nm2 amplitude error image
(Figure 3D) showed a veinlike texture between the nano-
5 particles. In the corresponding phase image (Figure 3F),
5
1
y 31, 200921/am9002 tntohaednsioespcfeaerrantitcublreeestws.Teahepenpceaoannroetrrdagsaintniinccotmhneatrtpaeshrtiaainsl,egciopmnhasagisseetsinccgoaolnofrbpetrooutsteheinde
AB on Juldoi: 10.10 otviercrleoystdh(i3effr5e,hr3eyn6dt)r.ionLcytaesrrobazcoytnimocneosamwnpditohcurtynhsdetsaA,lFliaMnnedtsipiml,vreeetrsawullliotciunnlgdaiinnmocppleaaarrrt-
CH Lorg | differencesinthephaseimages.Consideringthattheelec-
Rs. trodepositionbuffercontainedprimarilysilverandlysozyme,
Aac
Es. the phase-contrasting materials are most likely distinct
Sb FIGURE 2. Cyclic voltammograms of pure lysozyme (A) and the
RCE REhttp://pu lminysietotihzaylamnvooell-t.asImnilvmeeaorcgnhraacmnaosipesa,srfihtiovcewlenveoillnetacbmtlraomcpkoh,goarrnaemdsisssuswcoceleursetsiiorveneco(vBord)lteiandm.5mT0h%oe- rthegeisopnhseorifctahlepsreottrwusoiosnusbsotnanthceesb.laTdheesruergfiaocnesasruerproroupnodsinedg
On to be areas of lysozyme packed between the silver nano-
R F09 o gpruarmeslyasroezyinmiencfrreoamsipnagrlytAligihstsehrogwrany.aTshaediansihtieadlvbollatcakmlminoegirnampaortf particles. Similar grafting features have been observed in
ed by AIuly 2, 20 Bsh.oTwhenvaosltaadmamshoegdragmrayofli0n.e5.mMsilveracetatein50%methanolis AchFeMmpichaalsdeecpoonsittriaosnt iomfasgilevserofnasnurofpaacretsicalefstearntdheaecrleyclatrtoe-,
wnloaded on J napapnloiepda,rtaicrleedsfiollmutifoonr.mAefdteornathceosnusrtfaanctedocfthpeotbelnatdiaelswanads wsithioernefaorscuesdtatihneedorcgaatnhoicdmicaotveerriaplooteffntthiaelsdiulvreinrgnaelneocptraordtiecpleos-
Dosh andontotheelectrodesurface(36).
bli needles.Muchofthiscoatingwasremovedafterextensive
Pu washingwithdeionizedwater,butathincoatingremained Evaluation of the Lysozyme Activity within
Surface Coatings. The hydrolytic activity of lysozyme
onthemetalsurfaces(Figure3,left).Theevenlydispersed
andstronglyadherentcoatingwasnotobservedwhenany retained within electrodeposited coatings on blades and
of the control solutions were used in the electrophoretic needleswasinvestigatedthroughaninvitrocelllysisassay
deposition assay. A uniform coating was not seen when designed to mimic the normal use of the instruments.
mostofthelysozymewasdialyzedfromasilvernanoparticle Coatedbladesandneedleswereusedtomakeincisionsand
solutionpriortoelectrophoresisorwithsolutionsoffreshly punctures,respectively,intoagaroseinfusedwithM.lyso-
mixedlysozymeandsilveracetate,whichdidnotcontain deikticuscells.Withinminutes,zonesofclearingwereseen
anynanoparticles.Atranslucentcoatingwasobservedwhen atthecontactsite,whichwasindicativeofcelllysisdueto
only lysozyme was present in solutions, but the film was lysozyme hydrolytic activity (Figure 5). The spread of cell
easilywashedoffthebladesurface. lysis over time within the agar demonstrated that the
The coatings from lysozyme-silver nanoparticle elec- antimicrobialactivitynotonlywasretainedonthesurface
trodepositionsolutionsretainedlysozyme,asshownbyATR ofthebladesandneedlesbutalsowastransferredintothe
FT-IR (Figure 4). The spectrum of the coated blades was media.Bladessubjectedtoelectrodepositionusingasolution
identical to the spectrum of blades that were coated with containingonlylysozymedemonstratedaminimalzoneof
lysozymeonly,aswellastothespectrumofthelysozyme clearingafterincubationfor16h,incomparisontoblades
stock.Theresultsconfirmedthatnomajorperturbationsin coated with lysozyme-silver nanoparticles. Cell lysis was
thelysozymestructureoccurredasaresultofsilvernano- notobservedwithbladesandneedlessubjectedtoelectro-
particlesynthesisorelectrophoreticdeposition.Changesin phoreticdepositionusingsolutionscontainingsolublesilver
1556 VOL.1•NO.7• 1553–1560•2009 Ebyetal. www.acsami.org
A
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1, 2009am9002
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AB on Juldoi: 10.10 brpelhasapdseeec.itTmivheaelgyse.usAr(fEFaMcaenodafrFeth)aeairm2e0as0gheondwmwn2afirsmo1amgµelmewf2ta(tAso,crCoigm,haptn.ldeHteEei)dgahwntidstch2ai0nle0tbhnaemrsb2oi(nxBe,admD,aparleintaduodFfe).tihmTehaebgleaasmdaeprlesiut3ur0fda0ecae(nAsdha5on0wdnnBm)i,nafotmhrep1l1iµtumµdm2ea2enirmdroa2rg0e(0C.namnd2iDm),agaensd,
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FIGURE4.ATRFT-IRofbladesurfacesafterelectrodepositionusingeitherthelysozyme-silvernanoparticlesolution(topspectrum)ora
solutioncontainingonlylysozyme(bottomspectrum).Inset:Spectrumofthelyophilizedlysozymestock.
acetate (no nanoparticles) or only silver nanoparticles (re- the media, which transferred the antimicrobial properties
sultsnotshown).Consideringthatsilverdidnotinitiatecell oflysozymethroughoutthecutandpuncturesites.
lysis under these conditions, the higher activity of the Antimicrobial Range of Surgical Blade Coat-
lysozyme-silver nanoparticle coatings versus lysozyme ings. The M. lysodeikticus lysis assay demonstrated the
alone is attributed to either a more efficient deposition of antimicrobialactivityofthecoatings.However,thecon-
lysozymeontothemetalsurfacewhensilvernanoparticles tributionofsilvernanoparticlestotheantimicrobialactiv-
arepresentorthecapabilityoftheenzymetodiffusemore ityofthecoatingscannotbedeterminedusingthisassay.
rapidlyfromlysozyme-silvernanoparticlecoatings.Either Lysozyme is primarily bacterolytic toward Gram-type
way,theuniquenatureofthelysozyme-silvernanoparticle positive strains, whereas silver nanoparticles have been
coatingsallowedtheefficientreleaseofactiveenzymeinto showntohaveantimicrobialactivitytowardmanydiffer-
www.acsami.org VOL.1•NO.7• 1553–1560•2009 1557
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FIGURE5.Celllysisassaymeasuringtheantimicrobialactivityofcoatedbladesandneedles.Coatedblades(leftandmiddle)andneedles
(right)wereusedtomakeincisionsandpunctures,respectively,andthenplacedontopofagaroseinfusedwithM.lysodeikticuscells.The
topbladecontainsacoatingoflysozyme-silvernanoparticles,whilethelowerbladehasacoatingoflysozymeonly.Zonesofcelllysisare
seenattheincisionandpuncturesites,aswellassurroundingthebladesandneedlesafterincubationat37°Cfor30minand16h.
Table1. AntimicrobialActivityofCoatedBladesagainstBacterialandYeastStrains
logreductiona
strain 1.5h 3h inhibitiontypeb
5
5
1, 2009am90021 ABBaaciccniillelluutossbsaauncbtthetirrlaibscaiyslSyiterne 234...457908(((110...425850 234...997838(((100...765520 bbbaaacccttteeerrriiiccciiidddaaalll
y 321/ Candidaalbicans growth growth none
CH LAB on Julorg | doi: 10.10 EKPStsslaecebphushediryoeilmlcolhacoiopnacnaccseuouslflimuaoournreeisaucesens 1302....52606951((((1100....36268742 g303r...924o468wt(((h100...135403 nbnbaaooccnntteeeerriicciiddaall
ARacs. Staphylococcusepidermidis 0.47(0.55 0.43(0.24 bacteriostatic
Es.
RCE REShttp://pub preodainulotcgtaiorftneedrvuacilntuiceousnbaawntaidosntchaewlcvituihalabtaielidtcyoaaostfe[ltdohgeboslaufdbvecia]u.bltlTuehrceeeslslmsta(ancdfdeuarfmrdoLmd-e1)tvhbiaeetfiioonnriteiaieslx2fp4rooshmuirnetchturobeeaaticsooenaptawerdiathtbelcaoadsaest]eady-sb.[llaobdgTehosef.tSihneeehivtbhiaietbiotleenxctteyfllopsre(dcwfeutaasmilsbL.a-s1)edatoantimloge
On
R F09 o entmicrobialspecies.Todeterminetherangeofantimi- fresh media (1:10) and incubated for 24 h to determine if
AI20 crobialactivitydemonstratedbybothcomponentsofthe anyremainingviablecellscouldproliferateintheabsence
ed by uly 2, cstoraatiinngw, eerieghutsdeidffeirnentthebaacntetirmiailcrsotrbaiianlsaasnsadyos.neCoyaetaesdt oStfecrnoea,teadndblBa.dseusb.tiSli.saduidrenuso,tvKi.sipbnlyeugmroowniaafet,erBt.haenstuhbraccuils-
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bli spread-plating culture aliquots on solid media and enu- afteranextendedexposuretocells,(ii)coatedbladeswere
u
P meratingthecfu(Table1).Uncoated,etchedbladeswere abletoinhibitsubsequentgrowthofanyviablecellsthatmay
alsoincubatedinseparateculturesand,ineachcase,cells have attached to coated and uncoated areas of the blade
grewtohighcelldensity,ensuringthattheetchedstainless during the first incubation, and (iii) coated blades demon-
steeldidnotadverselyaffectthegrowth(resultsnotshown). strated a complete kill in the initial culture after 24 h. A.
Demonstratingeffectivebactericidalactivity,coatedblades baylyiwasoneexception,whereonesubculturegrewtohigh
reduced the cell viability by >3 log within 1.5 h for K. cell density. We conclude that the effect on A. baylyi was
pneumoniae,B.anthracisSterne,andB.subtilisandwithin bactericidal, but the potency of the coatings under our
3hforS.aureus.Alesserbiocidaleffectwasobservedwhen conditions was very close to the minimum bactericidal
A.baylyicellswereexposedtocoatedblades(∼3login3h). concentration,andgrowthintheonesubculturewasdueto
Growth inhibition lasted beyond 3 h because no visible standarddeviationorexperimentalerror.
growth was observed in cultures when incubated in the Aninitial,bactericidaleffectwasmeasuredforE.coli,but
presenceofcoatedbladesfor24h.Toassessifcoatedblades the strain was able to recover and turbid cultures were
retainedantimicrobialactivityafterexposuretoculturesand observedafter24hincubation.Previousworkdemonstrated
also inhibited the transfer of any viable cells on blade that silver nanoparticles were an effective antimicrobial
surfaces,bladeswereremovedfromtheculture,addedto agenttowardthisparticularstrainofE.coli(16,21).Wedid
freshmedia,andincubatedforanadditional24h.Withthe observehigherbactericidalactivitywhentheinitialnumber
five sensitive strains mentioned above, there was no ob- ofE.colicellswasreducedto104incultures(approximately
servedgrowthinthesubsequentcultures.Furthermore,the 3logreductioninviablecellswithin1.5h;datanotshown).
culturemediafromthefirst24hincubationwasdilutedinto Weconcludedthattheconcentrationofsilverionsreleased
1558 VOL.1•NO.7• 1553–1560•2009 Ebyetal. www.acsami.org
intotheculturebycoatedbladeswasbelowtheminimum homogeneous coatings on stainless steel surfaces. The
inhibitoryconcentrationforE.coliundertheconditionsof natureofthecoatingwassuchthatthemixtureoflysozyme
theantimicrobialassay.SimilartoE.coli,aninitial,bacte- and silver nanoparticles facilitated strong adsorption to A
ricidaleffectwasalsomeasuredforP.fluorescens,butthe stainless steel surfaces, yet the active components also
R
survivingpopulationrecoveredandpropagatedafter24h. diffusedfromthecoatingfollowingcontactwithmedia.The
T
WhenP.fluorescensculturesweretransferredandgrownon observations indicate that the physical properties of the
I
solidmediatoenumerateviablecells,coloniesoriginating coatingareinherenttolysozyme,wheretheproteinactsas C
fromcoatedbladecultureswerenearlyidenticalwithcolo- awater-solubleadhesiveandenablesrobustadsorptionof L
niesgrownfromuncoatedbladeculturesbutdidnotproduce the silver nanoparticles to the blade surface but will also E
thecharacteristicyellow-greenpyoverdinepigmentsthatare permitdiffusionofsilverionsorreleaseofthenanoparticles
normallyobservedwithrobustcells(37).Thecauseofthis themselves once incubated in an aqueous environment.
phenotype is not clear although it suggested that residual Hence, the effect could provide a persistent self-cleaning
silverdisruptedthepyoverdinesynthesisand/orexportfrom surfaceforthemedicalinstrumentsandallowtransferofthe
thecell. antimicrobialactivitytothecontactsurfaceduringuse(e.g.,
TheeffectofcoatedbladesonS.epidermidiswasconsid- diffusion into the surrounding flesh following an incision,
ered to be bacteriostatic. On average, culture viability did injection,orimplant).Withrespecttoimplanteddevices,the
notchangesignificantlyinviabilityduringthefirst3hafter integration of lysozyme within the composite may also
exposure to coated blades and did not grow to visible increasethebiocompatibilityofthecoatingandnothamper
turbidity after 24 h. Upon transfer of the blades in fresh tissuereconstruction,comparedtoothersilvercoatingsthat
5 media, cultures grew to a slight turbidity, suggesting that arecomposedsolelyofinorganicmaterial(39).Furthermore,
5
AB on July 31, 2009doi: 10.1021/am90021 bls2meouxl4saabetdlhcrleytsueoi.lcntdmTuohrataeheetbdeionsaifugacnsbbttteohsssreeetfiinqorilrelsucstieepnahnothomittcifebceneciudtocfelfityaaedu.tcraeSteWnd.sodehnbcpielonaiSndncd.eufieetrbrhpsmma,eidtitdeeeihniddrsemifttgoichiardreaolilatslwcsn.tuhtUgahletrdnuebdcdrwuoieettaironwthtinonhaaardegs-l ta(inon4hlporg0etet,iframeo4adfr1pfiyzea)i,adcadittcnciiiocvvoceenerapertasoaitnegefsagdirnniamnfagnosnigerttoetsuaphposlaolefordsattiteinpprcnaprltetiehlniisacesselayfnfttnooeitotoreshntddteersaahds.ninWesisrdnieahtmnitieolhdemneistddthpioseceltopamucyoldosaisyannir,ttdeuiifonuafuanggsrctetwrwthniueaeitrlsssr-l
CH Lorg | ctioonndaitniodngsroowftthheoafsSs.aeypsi,decromatieddisbbluatddesidinnhoitbcitoemdprleepterolydukicl-l improveitspotency.
Rs.
EAs.ac cells.Onceremovedfromexposuretocoatedblades,cells Acknowledgment.WethankKarenKellyattheUniver-
E RESp://pub recTohveerreedwaansdngoraepwp.arenteffectagainsttherepresentative sRietyseoafrcFhl,oErildeactrIonnteMrdiicsrcoipsclionpayryCoCreenLtearb,foGraiBnisovtiellceh,nFoLl,ofgoyr
RChtt fungal strain C. albicans. When cultures of this yeast were generating TEM images, Robert K. Nichols for helpful dis-
On
AIR F2009 o ecuxlptuorseesdctoontcaoiantiendgubnlacdoeast,edthbelyadgerse.wInaotutrhperseavmioeusrsattuedays, ceursfsoiornassosnisetlaenctcreopinhoAreFtMicdteepcohsnitiiqoune,as.ndTKhiesnnweothrkStwraawshseucpk--
ed by uly 2, tshioenesffaegcatiivnesnteCs.saolfbliycsaonzsymincer-esaislveedrwnahneonplayrstoiczleymsuespweans- pMoarnteudfatchturoriungghDfiurnecdtionrgatfero(AmFRthLe/RAXi)raFnodrcteheMJoaitnetriSaclsieanncde
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wnloaed on rweemreovceodnsfrisotmentthweistholuotuiornp(r2e1v)i.ouTshefinredsiunlgtss ibnecthaiussestutdhye TPerocjhencotlCoogydeOAfAfi0ce6CfBoTr0C0h8e(mIlyicaalElaanshdviBlii,oJleongniciafelrDBeefceknesre,
Dosh andStephenLee,ProgramManagers).
bli coatings were created from nanoparticle suspensions that
u
P containedaconsiderablyhigherconcentrationoflysozyme
REFERENCESANDNOTES
and,asaresult,werelesseffectiveagainstC.albicanscells.
(1) Hetrick,E.M.;Schoenfisch,M.H.Chem.Soc.Rev.2006,35,780–
789.
CONCLUSIONS (2) Babu,R.;Zhang,J.;Beckman,E.J.;Virji,M.;Pasculle,W.A.;Wells,
A.Biomaterials2006,27,4304–4314.
Ourstudiesdemonstratedthatcompositeantimicrobial
(3) Hardes,J.;Ahrens,H.;Gebert,C.;Streitbuerger,A.;Buerger,H.;
coatings can be generated using methods for lysozyme- Erren,M.;Gunsel,A.;Wedemeyer,C.;Saxler,G.;Winkelmann,
mediatedsilvernanoparticlesynthesisandelectrophoretic W.;Gosheger,G.Biomaterials2007,28,2869–2875.
(4) Ong,S.-Y.;Wu,J.;Moochhala,S.M.;Tan,M.-H.;Lu,J.Biomaterials
deposition. The coating employs two different biocidal
2008,29,4323–4332.
mechanisms:theantimicrobialactivityofsilverionsandthe (5) Dowsett,C.Nurs.Stand.2004,19,56–60.
muramidase activity of lysozyme. Coatings that integrate (6) Strohal,R.;Schelling,M.;Takacs,M.;Jurecka,W.;Gruber,U.;
different biocidal mechanisms are of particular interest Offner,F.J.Hosp.Infect.2005,60,226–230.
(7) Silver,S.FEMSMicrobiol.Rev.2003,27,341–353.
becausetheycanminimizetheselectionandproliferation
(8) Lansdown,A.B.G.Curr.Prob.Derm.2006,33,17–34.
ofresistantstrains.Silvernanoparticleshavealreadybeen (9) Schierholz,J.M.;Lucas,L.J.;Rump,A.;Pulverer,G.J.Hosp.Infect.
shown to be effective in suppression of a wide range of 1998,40,257–262.
(10) Baker,C.;Pradhan,A.;Pakstis,L.;Pochan,D.J.;Shah,S.I.J.
pathogens,especiallyagainststrainsresistanttomainstream
Nanosci.Nanotechnol.2005,5,244–249.
antibiotics(6,38).Whilethecontributionoflysozymehas (11) Pal,S.;Tak,Y.K.;Song,J.M.Appl.Environ.Microbiol.2007,73,
been shown to minimally contribute to the antimicrobial 1712–1720.
(12) Kim,Y.H.;Lee,D.K.;Cha,H.G.;Kim,C.W.;Kang,Y.S.J.Phys.
activityofthecomposite(21),theenzymewasessentialto
Chem.C2007,111,3629–3635.
electrophoreticdepositionandpromotedtheformationof (13) Kong,H.;Jang,J.Langmuir2008,24,2051–2056.
www.acsami.org VOL.1•NO.7• 1553–1560•2009 1559
(14) Lee,D.;Cohen,R.E.;Rubner,M.F.Langmuir2005,21,9651– (28) Spoerke,E.D.;Stupp,S.I.Biomaterials2005,26,5120–5129.
9659. (29) Fleming,A.Proc.R.Soc.London,Ser.B1922,93,306–317.
(15) Li,Y.;Leung,P.;Yao,L.;Song,Q.W.;Newton,E.J.Hosp.Infect. (30) Ireland,J.A.;Hanna,P.C.Infect.Immun.2002,70,5870–5872.
E 2006,62,58–63. (31) Juni,E.;Janik,A.J.Bacteriol.1969,98,281–288.
L (16) Marini,M.;Niederhausern,S.D.;Iseppi,R.;Bondi,M.;Sabia,C.; (32) Barton,A.C.;Collyer,S.D.;Davis,F.;Gornall,D.D.;Law,K.A.;
Toselli,M.;Pilati,F.Biomacromolecules2007,8,1246–1254. Lawrence,E.C.;Mills,D.W.;Myler,S.;Pritchard,J.A.;Thomp-
C
(17) Sambhy,V.;MacBride,M.M.;Peterson,B.R.;Sen,A.J.Am.Chem. son,M.;Higson,S.P.Biosens.Bioelectron.2004,20,328–337.
I Soc.2006,128,9798–808. (33) PiresdeCastro,C.S.;RodriguesSouzaDe,J.;BlochJunior,C.
T (18) Vigneshwaran, N.; Kathe, A. A.; Varadarajan, P. V.; Nachane, J.Inorg.Biochem.2003,94,365–371.
R R.P.;Balasubramanya,R.H.Langmuir2007,23,7113–7117. (34) Socrates,G.Infraredandramanchracteristicgroupfrequencies.Tables
(19) Wang,Q.;Yu,H.;Zhong,L.;Liu,J.;Sun,J.;Shen,J.Chem.Mater. andcharts,3rded.;JohnWileyandSons,Ltd.:Chichester,U.K.,2001.
A 2006,18,1988–1994. (35) Lin,S.;Lee,C.K.;Lee,S.Y.;Kao,C.L.;Lin,C.W.;Wang,A.B.;
(20) Zhang,Y.;Peng,H.;Huang,W.;Zhou,Y.;Zhang,X.;Yan,D.J. Hsu,S.M.;Huang,L.S.Cell.Microbiol.2005,7,1763–1770.
Phys.Chem.C2008,112,2330–2336. (36) Voccia,S.;Ignatova,M.;Jerome,R.;Jerome,C.Langmuir2006,
(21) Eby,D.M.;Schaeublin,N.M.;Farrington,K.E.;Hussain,S.M.; 22,8607–8613.
Johnson,G.R.ACSNano2009,3,984–994. (37) Meyer,J.-M.Arch.Microbiol.2000,174,135–142.
(22) Pellegrini,A.;Thomas,U.;vonFellenberg,R.;Wild,P.J.Appl. (38) Atiyeh,B.S.;Costagliola,M.;Hayek,S.N.;Dibo,S.A.Burns2007,
Bacteriol.1992,72,180–187. 33,139–148.
(23) Pana´eˇek, A.; Kv´ıtek, L.; Prucek, R.; Kola´rˇ, M.; Vecˇerˇova´, R.; (39) Meyers,S.R.;Khoo,X.;Huang,X.;Walsh,E.B.;Grinstaff,M.W.;
Pizu´rova´,N.;Sharma,V.K.;Neveˇcˇna,T.;Zborˇil,R.J.Phys.Chem. Kenan,D.J.Biomaterials2009,30,277–286.
B2006,110,16248–16253. (40) Silver,S.;Phung,L.T.;Silver,G.J.Ind.Microbiol.Biotechnol.2006,
(24) Lim,S.H.;Wei,J.;Lin,J.;Li,Q.;Kuayou,J.Biosens.Bioelectron. 33,627–634.
2005,20,2341–2346. (41) U.S.FoodandDrugAdministration,CenterforFoodSafetyand
(25) Luo,X.L.;Xu,J.J.;Du,Y.;Chen,H.Y.Anal.Biochem.2004,334, AppliedNutrition,OfficeofPremarketApproval.AgencyResponse
284–289. LetterGenerallyRegardedasSafe(GRAS)NoticeNo.GRN000064;
(26) Wu,L.Q.;Lee,K.;Wang,X.;English,D.S.;Losert,W.;Payne, April2,2001.
5
5 G.F.Langmuir2005,21,3641–3646.
1
1, 2009am9002 (27) Zhao,G.;Xu,J.J.;Chen,H.Y.Anal.Biochem.2006,350,145–150. AM9002155
y 321/
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1560 VOL.1•NO.7• 1553–1560•2009 Ebyetal. www.acsami.org