Plasmon Resonance of Silver Nanoparticles as a Method of Increasing Their Antibacterial Action

18 Pages·2017·3.33 MB·English

antibiotics
Article
Plasmon Resonance of Silver Nanoparticles as a
Method of Increasing Their Antibacterial Action
AlexanderYu.Vasil’kov1,RuslanI.Dovnar2,* ID,SiarheiM.Smotryn2,NikolaiN.Iaskevich2
andAlexanderV.Naumkin1 ID
1 NesmeyanovInstituteofOrganoelementCompounds,RussianAcademyofSciences,28Vavilovst.,
Moscow119991,Russia;[email protected](A.Y.V.);[email protected](A.V.N.)
2 GrodnoStateMedicalUniversity,80GorkySt.,Grodno230009,Belarus;[email protected](S.M.S.);
[email protected](N.N.I.)
* Correspondence:[email protected];Tel.:[phone]
(cid:1)(cid:2)(cid:3)(cid:1)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:1)
(cid:1)(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)
Received:14July2018;Accepted:21August2018;Published:22August2018
Abstract: In this article, a series of silver-containing dressings are prepared by metal-vapor
synthesis(MVS),andtheirantibacterialpropertiesareinvestigated. Theantibacterialactivityofthe
dressingscontainingsilvernanoparticles(AgNPs)againstsomeGram-positive,andGram-negative
microorganisms(Staphylococcusaureus,Staphylococcushaemolyticus,Pseudomonasaeruginosa,Klebsiella
pneumoniae, Escherichia coli, Moraxella spp.) has been determined. Based on the plasmon resonance
frequencyofthesenanoparticles,thefrequencyoflaserirradiationofthedressingwaschosen.Thegauze
bandageexaminedshowedpronouncedantibacterialproperties,especiallytoStaphylococcusaureus
strain.When470nmlaserradiation,withapowerof5mW,wasappliedfor5min,4hafterinoculating
thePetridish,andplacingabandagecontainingsilvernanoparticlesonit,theantibacterialeffectof
thelattersignificantlyincreased—bothagainstGram-positiveandGram-negativemicroorganisms.
Thestructureandchemicalcompositionofthesilver-containingnanocompositewerestudiedby
transmissionelectronmicroscopy(TEM),X-rayphotoelectronspectroscopy(XPS)andextendedX-ray
absorptionfinestructure(EXAFS).ThesynthesizedAgNPsdemonstratenarrowandmonomodal
particlesizedistributionwithanaveragesizeof1.75nm. AtomsofmetalinAg/bandagesystemare
mainlyinAg0state,andtheoxidizedatomsareintheformofAg-Ag-Ogroups.
Keywords: antibacterialeffect;laserirradiation;metal-vapourmethod;silvernanoparticles;TEM;
XPS;EXAFS
1. Introduction
Thewidespreaduseofantibioticsforthetreatmentandpreventionofbacterialdiseasesleadsto
asignificantincreaseintheantibacterialresistanceofmicroorganismsthroughitsacquisitioneither
throughexogenousresistancegenesorchromosomalmutations[1]. Thisstimulatesnotonlythesearch
fornewantibacterialdrugs,butalsopossiblealternativestothelatter[2,3]. Thescientistsaretaskedto
findsubstancesthateffectivelyactsimultaneouslyonGram-positive,Gram-negativemicroorganisms
andfungi,whichareindependentoftheantibioticresistanceofthemicroorganism.
Silveranditscompoundshavebeenusedinmedicinesinceancienttimes. Massapplicationof
silverpreparationsbeganintheseventiesofthe19thcentury[4,5]. Sincethen,numerousconfirmations
ofantiviral,antibacterial,andimmunomodulatingactivityofsilverpreparationshavebeenreceived[6].
Withtheinventionofantibiotics,whichhavemorepronouncedantibacterialproperties,interestin
thetherapeuticpropertiesofsilverdramaticallydecreased. However,widespreaduseofantibiotics
revealedbytheendofthe20thcenturyanumberoftheirshortcomingsandsilverpreparationswere
againactivelystudiedandused[7].
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The mechanism of antimicrobial effect of silver was carefully studied previously. It has now
beenestablishedthatsilverionsareselectivelytoxicwithrespecttoprokaryoticmicroorganismswith
aweakeffectoneukaryoticcells[6],includingcomparativelyminimaltoxicitytomammaliancells[8].
Thisisduetothefactthattheconcentrationofsilverionsorsilvernanoparticlesnecessaryforthe
deathofprokaryoticcells(bacteria)ismuchlowerthantheconcentrationthatcausesthedeathof
eukaryoticcells,includingcellsofthehumanbody[9].
Itshouldbenotedthatnewprospectsfortheuseofsilverinmedicineareopenedinconnection
with the development of nanotechnology, an interdisciplinary field of science that deals with the
creation, production and application of structures, devices and systems ranging in size from 1 to
1000nm,althoughinpracticetheyusethesizesrangingfrom1to100nmmoreoften[10]. Itisproved
thatthemetalnanoparticleshaveuniqueproperties,oftendifferingfromthatofthesolidmetal[10].
Thisisduetothefactthatthesurface/bulkenergyratioofnanoparticlesismuchlargerthanthatof
compactmetal[11].
As applied to medicine, this means that the nature of the interaction of a nanoparticle with
a bacterium or fungus is significantly different from the impact of a compact metal on them and
probablyenhancestheirbactericidalorfungicidalactivity[10].
Localized surface plasmon resonance is an optical phenomenon that is generated when light
interactswithconductivenanoparticlesthataresmallerthantheincidentwavelength[12]. Fromthe
pointofviewofantibacterialpropertiesof(silvernanoparticles)AgNPs,itisinterestingtostudyhow
thesepropertieschangewhenplasmonresonanceeffectoccurs.
Oneoftheecologicalandeffectivemethodsforproducingmono-andbi-metallicnanoparticles
andmaterialsbasedonthemisthemetal-vaporsynthesis(MVS).Themethodwasusedforpreparation
ofsilver-containingcompositematerialsformedicalapplications[13]. ItisassumedthatMVSwill
be effective for the modification of wound dressings prepared from natural or synthetic materials
withAgNPs.
Inthisregard,thewideintroductionofdressingscontainingAgNPscorrectlycombinedwiththe
plasmonresonanceeffectcanplayasignificantroleinimprovingtreatmentofpurulentwoundsinthe
eraofincreasingantibioticresistanceofmicroorganisms.
Theaimofthisresearchistostudytheantibacterialeffectofanewdressingmaterial,basedon
gauzebandagecontainingAgNPspreparedbyMVS,andchangingthiseffectundertheinfluenceof
laserradiation.
2. Results
Thestructureandchemicalcompositionofthesilver-containingnanocompositewerestudiedby
transmissionelectronmicroscopy(TEM),X-rayphotoelectronspectroscopy(XPS)andextendedX-ray
absorptionfinestructure(EXAFS).
TheTEMmicrographofthecottonfibreisshowninFigure1. Thephotographshowsamorphous
extendedstructurewithadiameterintherangeof16–25nm, anddarkcrystallinenanostructures,
thedensityofwhichishigherthanthatofcotton. Theenlargedimageoftheregionsoforderedatoms
onasurfacemarkedwithasquareinFigure1isshowninFigure2.
TheEDSspectraobtainedfromdarknanostructures(notshown)containthecharacteristicline
AgLα=2.98keV,whichallowsattributedarknanostructurestoAgNPs. MostofAgNPsareinthe
formofchainsandagglomerates,whichisacharacteristicfeatureofthesystemspreparedbyMVS.
Theparticlesizedistributionisnarrowandmonomodal. Theaverageparticlesizeis1.75±0.25nm.
SimulationoftheelectrondiffractionforthegroupoftheatomsselectedinFigure2isshownin
Figure3. ItisseenfromFigures2and3thatgroupsofatomsformfaceswithinterplanardistances
d1andd2. Calculatingtheratiod1/d2givesavalueof1.09. Theanglebetweend1andd2is52◦.
Thevalues1.09and52◦ indicatethepresenceoffaces(111)and(200)ofaface-centeredcubic(fcc)
structureinsilverparticles(foranidealfccstructure: d(111)/d(200)=1.15,theangleis55◦).
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Figure 1. Transmission electron microscopy (TEM) micrograph of Ag-cotton system.
Figure1.Transmissionelectronmicroscopy(TEM)micrographofAg-cottonsystem.
Based on the synthesis method and sample storage conditions, three models of the chemical
compositionoftheparticlesurfacecanbeproposed: Ag0,Ag+,andAg0 +Ag+. Withahighdegree
of probability, the mixed composition in crystalline form can be excluded from the consideration.
Otherwise,“doublereflexes”shouldbeobservedinFigure3. Thefcclatticeconstants(a)forAg0and
Ag Oare4.08Åand4.76Å,respectively. Calculationofabytheformulaa=d(hkl)×(h2+k2+l2)1/2,
2
whereh=1,k=1,l=1;d(hkl)=2.3Å,givesavalueof3.98Å.Consequently,thereisreasontobelieve
thatthesurfaceofthecrystallineparticlesconsistsofmetallicsilver. However,presenceofAg+statein
amorphousformcannotbeexcluded.
Figure4showsthesurveyspectrumofAgblack. Alongwiththepeakscharacteristicofsilver
atomstherearepeaksofimpuritycarbonandoxygenatoms.
ThedeterminationofthechemicalstateofsilveratomsinnanoparticlesbytheXPSmethodis
acomplextask. Thisisduetothefactthatthespectralcharacteristicsofthemetalparticlesandthe
oxideparticlesarefairlyclose. AccordingtoNISTXPSDatabase[14]thebindingenergiesoftheAg
3d peak for Ag, Ag O and AgO are in the ranges 367.9–368.4, 367.7–368.4, and 367.3–368.1 eV,
5/2 2
respectively. OneofthesolutionstothisproblemistheuseoftheAugerparameter. However,asarule,
theconcentrationofsilvernanoparticlesinmaterialsislow, whereastheregistrationoftheAuger
spectrumrequiresasignificantlylongertimethantherecordingofthephotoelectronspectrum,which
canleadtothereductionofsilverfromoxidesundertheactionofX-rayirradiation[15].
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Ag(111)

Ag(200)

Figure 2. An enlarged image of the regions of ordered atoms on a surface, marked with a square in
Figure2. Anenlargedimageoftheregionsoforderedatomsonasurface,markedwithasquarein
Figure 1.
Figure1.
The EDS spectra obtained from dark nanostructures (not shown) contain the characteristic line
AAgLnαot =h e2r.9a8p kperVoa, cwhhmicahy ablleowbass aetdtroibnucteo ndtarrokll endandoisfftreurecntutiraels cthoa ArggiNngP,si. nMwohsti cohf sAegpNarPast iaorne oinf stihgen als

fromforrmeg oiof ncshawinitsh angdo oadggalnomdeproatoers,c wonhdicuhc itsi vai tcyhaisrapctoesrsisibtilce f.eIaftuthree oref gthioe nsyhsatesmasm preetpalalriecdc obny dMuVctSi.v ity
andTihsei npagrtoioclde seilzeec dtriisctarilbcuotinotna cist nwairtrhowth aensda mmopnleomhoodldael.r T, thhee anveitradgoee psanrtoictlea cscizuem isu 1la.7t5e ±a 0c.2h5a nrgme. due
to the emSiimssuiloantioonf osfe tchoen edleacrtyroenl edcitfrforancsti.onIn focro tnhter agsrto,uipn oaf trheeg iaotonmws istehlepcoteodr inco Fnigduurcet i2v iist ys,haowpno sinit ive
Figure 3. It is seen from Figures 2 and 3 that groups of atoms form faces with interplanar distances
chargeisusuallyaccumulated,thevalueofwhichdependsonthesecondaryemissioncoefficientand
d1 and d2. Calculating the ratio d1/d2 gives a value of 1.09. The angle between d1 and d2 is 52°. The
conductivity,anditleadstothedisplacementofphotoelectronpeaksintheregionofhighbinding
values 1.09 and 52° indicate the presence of faces (111) and (200) of a face-centered cubic (fcc)
energies. WhenapositivebiasvoltageU isapplied,thestrayelectronsflowtothesamplesurface
structure in silver particles (for an ideal fbcc structure: d(111)/d (200) = 1.15, the angle is 55°).
is increased and contributes to the compensation of the surface charge. In this case, narrowing of
Based on the synthesis method and sample storage conditions, three models of the chemical
thephotoelectronpeaksandtheirshifttowardhigherbindingenergiesareobserved. Photoelectron
composition of the particle surface can be proposed: Ag0, Ag+, and Ag0 + Ag+. With a high degree of
peakscorrespondingtoregionswithgoodconductivityshouldbeshiftedbyU theamountofapplied
probability, the mixed composition in crystalline form can be excluded frobm the consideration.
biasvoltage,whilefromareaswithaworseconductivitybyasmalleramount. Withanegativebias
Otherwise, “double reflexes” should be observed in Figure 3. The fcc lattice constants (a) for Ag0 and
voltAagge2O,t hareefl 4u.0x8 sÅtr aanyde l4e.7ct6r oÅn, sredsepcercetiavseelsy,. iCtalelcaudlsattioonin ocfr ae absye tihnec fhoarmrguinlag ao =n dn(ohnk-l)c o×n (dh2u +c tkin2 +g lr2e)1g/2i,o ns
andwahnerien chr e=a 1s,e ki n= t1h, el =in 1te; rdv a(hlkbl)e t=w 2e.3e nÅp, geiavkess cao vrraeluspe oonf d3i.9n8g Åto. Csiognnsaeqlsuefrnotlmy, tthheerec oins dreuacstoinn gtoa nd
nonb-ceolinevdeu cthtiantg threeg siuornfsa.ceIn otfh tihsec carsyes,ttahlleinseig pnaarltifcrloems ctohnesicsotsn dofu mctievtaellriecg siiolvnemr. uHsotwalesvoebr,e psrheisfetnedceb oyf U .
b
ATga+ ksitnatge iinnt oamacocropuhnotutsh feordmif fcearnennocet bine tehxecleuldeecdtr.i c alconductivityofmetallicsilveranditsoxides,we
attemptedtoseparatesignalsfromregionscontainingsilveratomsinametalstateandotherchemical
statesbycontrolleddifferentialcharging,changingthepotentialonthesampleholder. Thisapproach
iswidelyusedtodeterminethepresenceofdifferentphasesinasample[16–21].
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(111)
(-111)
d
1
(200)
d
2 (-200)
(1-1-1) (-1-1-1)

Figure 3. Simulation of the electron diffraction pattern for the group of atoms circled in Figure 2 and
Figure3.SimulationoftheelectrondiffractionpatternforthegroupofatomscircledinFigure2and
Antibioticass 2s0ig18n,m 7,e xn t for face-centered cubic (fcc) structure. 6 of 19
assignmentforface-centeredcubic(fcc)structure.
Figure 4 shows the survey spectrum of Ag black. Along with the peaks characteristic of silver
atoms there are peaks of impurity carbon and oxygen atoms.
Ag 3d
2
u.
a.
y,
nsit O 1s C 1s
e Ag 3p
nt
I
1 O KVV
Ag MNN
Ag 4sAg 4p
C KVV
1000 800 600 400 200
Binding Energy, eV

Figure4.X-rayphotoelectronspectroscopy(XPS)surveyspectrumofAgblack.
Figure 4. X-ray photoelectron spectroscopy (XPS) survey spectrum of Ag black.
The determination of the chemical state of silver atoms in nanoparticles by the XPS method is a
complex task. This is due to the fact that the spectral characteristics of the metal particles and the
oxide particles are fairly close. According to NIST XPS Database [14] the binding energies of the Ag
3d5/2 peak for Ag, Ag2O and AgO are in the ranges 367.9–368.4, 367.7–368.4, and 367.3–368.1 eV,
respectively. One of the solutions to this problem is the use of the Auger parameter. However, as a
rule, the concentration of silver nanoparticles in materials is low, whereas the registration of the
Auger spectrum requires a significantly longer time than the recording of the photoelectron
spectrum, which can lead to the reduction of silver from oxides under the action of X-ray irradiation
[15].
Another approach may be based on controlled differential charging, in which separation of
signals from regions with good and poor conductivity is possible. If the region has a metallic
conductivity and is in good electrical contact with the sample holder, then it does not accumulate a
charge due to the emission of secondary electrons. In contrast, in a region with poor conductivity, a
positive charge is usually accumulated, the value of which depends on the secondary emission
coefficient and conductivity, and it leads to the displacement of photoelectron peaks in the region of
high binding energies. When a positive bias voltage Ub is applied, the stray electrons flow to the
sample surface is increased and contributes to the compensation of the surface charge. In this case,
narrowing of the photoelectron peaks and their shift toward higher binding energies are observed.
Photoelectron peaks corresponding to regions with good conductivity should be shifted by Ub the
amount of applied bias voltage, while from areas with a worse conductivity by a smaller amount.
With a negative bias voltage, the flux stray electrons decreases, it leads to increase in charging on
non-conducting regions and an increase in the interval between peaks corresponding to signals from
the conducting and non-conducting regions. In this case, the signal from the conductive region must
also be shifted by Ub.
Taking into account the difference in the electrical conductivity of metallic silver and its oxides,
we attempted to separate signals from regions containing silver atoms in a metal state and other
chemical states by controlled differential charging, changing the potential on the sample holder. This
approach is widely used to determine the presence of different phases in a sample [16–21].
To discriminate regions with different conductivities, or other words with different chemical
states, a bias voltage Ub = ±7 V was supplied to the sample holder. Figure 5 shows the Ag 3d spectra
of the Ag black measured at different bias voltage Ub applied to the sample holder. It is seen that
binding energy of the Ag 3d5/2 and Ag 3d3/2 peaks is slightly depends on Ub. Table 1 presents the
characterization of the Ag 3d spectra. The full widths at high maximum (FWHM) are rather different
Antibiotics2018,7,80 6of18
To discriminate regions with different conductivities, or other words with different chemical
states,abiasvoltageU =±7Vwassuppliedtothesampleholder. Figure5showstheAg3dspectra
b
oftheAgblackmeasuredatdifferentbiasvoltageU appliedtothesampleholder. Itisseenthat
b
binAdntiinbigotiecsn 2e0r18g,y 7, ox ftheAg3d andAg3d peaksisslightlydependsonU . Table1prese7n otf s19t he
5/2 3/2 b
characterizationoftheAg3dspectra. Thefullwidthsathighmaximum(FWHM)areratherdifferent
as well. Both the binding energies of the Ag 3d peaks and their FWHM values indicate that the
aswell. BoththebindingenergiesoftheAg3dpeaksandtheirFWHMvaluesindicatethatthespectra
consptaeicntrsao cmonetsatiant essomweit hstadtiefsf ewreintht cdoifnfedruecntti vciotniedsu.cCtiovnitsiieds.e rCinongstihdaetritnhge trheacto trhdein rgectohredAingg tOhea nAdg2AOg O
2
and AgO spectra is accompanied by the surface charging [22,23], one can assume the presence of the
spectraisaccompaniedbythesurfacecharging[22,23],onecanassumethepresenceoftheAg+and/or
Ag+ and/or Ag2+ state in the Ag black. To determine the characteristics of the Agδ+ state a subtraction
Ag2+ state in the Ag black. To determine the characteristics of the Agδ+ state a subtraction of the
of the spectrum measured at Ub = 7 V from the spectrum measured at Ub = −7 V was performed
spectrummeasuredatU =7VfromthespectrummeasuredatU =−7Vwasperformedunderthe
under the condition of btheir best coincidence in the high-energy rebgion. The difference spectrum is
conditionoftheirbestcoincidenceinthehigh-energyregion. Thedifferencespectrumispresentedin
presented in Figure 5. The binding energies of the Ag 3d5/2 and Ag 3d3/2 peaks 367.73 and 373.71 eV
Figure5. ThebindingenergiesoftheAg3d andAg3d peaks367.73and373.71eVcorrespondto
correspond to Ag2O state [22]. The relative5 /in2tensity of th3i/s2 state is no less than 0.27. This estimate is
Ag Ostate[22]. Therelativeintensityofthisstateisnolessthan0.27. Thisestimateisbasedonthe
b2ased on the fact that the spectrum measured at Ub = 7 V may contain Ag2O state.
factthatthespectrummeasuredatU =7VmaycontainAg Ostate.
b 2
Ag 3d
5/2
Ag 3d
4 3/2
u.
a. 3
y,
t
si
n 2 1
e
nt
I
2
1
3
0 4
378 376 374 372 370 368 366 364
Binding Energy, eV

Figure5.TheAg3dphotoelectronspectraofAgblackmeasuredatdifferentU =+7(1),−7(2),0V(3),
b
anFtidgiuffreer e5n. Tcehes pAegct 3rudm ph(o2)to–(e1le).ctTrohne ssppeeccttrraa oafr eAcgo rbrleacctke dmfeoarsuUred. at different Ub = +7 (1), −7 (2), 0 V
b
(3), ant difference spectrum (2)–(1). The spectra are corrected for Ub.
Table1.Bindingenergiesandfullwidthsathighmaximum(FWHMs)measuredbyXPS.
Table 1. Binding energies and full widths at high maximum (FWHMs) measured by XPS.
Ub Ub AAgg3 3dd5/52/2,, eeVV AgA 3gd33d/2,3 e/2V, eVAg 3dA5/2g F3WdH5/2MF,W eVH M,eV
0 V 368.17 374.17 1.3
0V 368.17 374.17 1.3
−7 V 368.09 374.11 1.4
−7V 368.09 374.11 1.4
+7 V 368.21 374.21 1.5
+7V 368.21 374.21 1.5
(−7V()−–7(+ V7)–V(+)7 V) 336677.7.733 3733.7731. 71 1.3 1.3
The C 1s and O 1s spectra of Ag black show a strong dependence on Ub (Figure 6) which
indTichaeteC t1hsata nad laOrg1es psparetc torfa coafrAbognb alancdk oshxyogwena sist rohnasg ldoewp ecnodnednuccetiovnityU. bIt( Fsihgouurled6 b)ew nhoictehdi ntdhaicta te
thabtinadlianrgg eenpearrgtyo focf athrbeo mnaainnd Co 1xsy gpeenaki smheaassluorwedc aotn Udub c=t i+v7i tVy. iIst 2s8h4o.u77ld ebVe, annodte cdotrhreastpboinnddsin tgo ethnaetr gy
ofuthseedm foari nchCar1gse preefaekremnceea [s1u4r]e. d atUb =+7Vis284.77eV,andcorrespondstothatusedforcharge
reference[14].
Itfollowsthatsomeofthecarbonatomshavegoodconductivity,orinotherwords,isinclose
contact with silver atoms. To estimate this value, we use the fitting the C 1s spectrum measured
atU = −7V,whenthebestseparationoftheelectronemissionfromregionswithgoodandpoor
b
conductivityisrealized. Whenthespectrumwasfittedwithsomecomponents,tworestrictionswere
imposed: Thewidthofthelow-energypeakshoulddescribethelow-energysidebythebestway,and
Antibiotics 2018, 7, x 8 of 19
4
O 1s C 1s
3
u.
a.
y, 2 1
sit
n
e
Antibiotics2018,7,80 Int 1 2 7of18
3
theenergyintervalbetweenthelow-energypeakandthenextshouldnotbelessthanthatatU =+7V.
0 b
Thesecondrestrictionisimposedbecauseofthepossiblemanifestationofdifferentialchargingfor
540 535 530 295 290 285 280
regionscontainingC-Ogroups. Thefractionofsuchcarbonatomsis0.47. Asimilarvalueof0.48was
obtainedforthespectrummeasuredatU B=in0dVin,wg hEenreeragsyf,o erVthespectrummeasuredatU =+7Vit
b b
is0.77. Thisisduetoconductivityinducedwiththestrayelectrons,whichmakeconductingregions

thatarenotindirectcontactwithsilvernanoparticles. Figure7showsfittingthecorrespondingC1s
AntFibiigotuicrse 2 061. 8T, 7h,e x C 1s and O 1s photoelectron spectra of Ag black measured at different Ub = +7 (1),8 − o7f 19
spectra,andTable2containscorrespondingdata.
(2), 0 V (3).
4
It follows that some of the carbon atoms have good conductivity, or in other words, is in close
contact with silver atoms. To estimOa te1 sthis value, we use the fCitt i1nsg the C 1s spectrum measured at Ub
= −7 V, when the best separation of the electron emission from regions with good and poor
3
conductivity is realized. When the spectrum was fitted with some components, two restrictions were
imposed: The width ou.f the low-energy peak should describe the low-energy side by the best way,
=a n+d7 t hVe. eTnheerg sye icnotnedrv arsity, a.le sbter2tiwctieoenn tihs ei mlopwo-seende rbgeyc paueaske1 aonf dt hthee pnoesxsti bshleo umlda nniofet sbtea tlieosns tohfa nd itfhfearte antt Uiabl
charging for regions cnontaining C-O groups. The fraction of such carbon atoms is 0.47. A similar
e
value of 0.48 was obtIntain1ed for the spectrum measu2red at Ub = 0 V, whereas for the spectrum
measured at Ub = +7 V it is 0.77. This is due to conductivity induced with the stray electrons, which
make conducting regions that are not in direct cont3act with silver nanoparticles. Figure 7 shows
fitting the corresponding C0 1s spectra, and Table 2 contains corresponding data.
It should be noted that th5e4 f0itting5 3th5e C 513s0 spect2ra9 5with2 s9o0me c2o8m5pon2e8n0ts measured at Ub = −7
and 0 V, presented in Figure 7 is largely conditional and do not reflect the real relative
Binding Energy, eV
concentrations of COx groups. At the same time, applying a positive bias voltage Ub = +7 V

practically compensates the surface charging, and Figure 7 (+7 V) reflects the real relative
Figure6.TheC1sandO1sphotoelectronspectraofAgblackmeasuredatdifferentU =+7(1),−7(2),
b
conce0nVFtri(ag3ut)i.roen 6s. oThf eC CO 1x sg arnodu pOs 1. s photoelectron spectra of Ag black measured at different Ub = +7 (1), −7
(2), 0 V (3).
5
6 It follows that some of the carbon atoms have good conductivity, or in other words, is in close
6 +7 V
contact w-7 iVth silver atoms. To estimate 5this 0v Value, we use the fitting the C 1s4 spectrum measured at Ub
Intensity, a.u.c=imo 24n−p7do usVecdt, i:vw iTthyhe ein sw rteihdaelt ihzb eoedsf .t t Whseeh pleoanwr ath-teieon snepIntensity, a.u. rgeoc234yft rptuhemea k we lseahcsot fruiotltnde dde emwsiictsrhsiib soeon mt hfere o clmoomw rp-eeoIntensity, a.u.gnnieoe23rnngstys ,ws tiwditeho brgeyos totrhdiec taibonendsst wwpoeaoryer,
1 1
and the energy interval between the low-energy peak and the next should not be less than that at Ub
0 0 0
= +72 94V.2 92Th29e0 s28e8co28n6d2 8r4es28t2ric28t0ion is impo29s4ed29 2b2e90ca2u88se2 8o6f 28t4he28 2po28s0sible mani2f9e4st2a92tio29n0 2o8f8 d28i6ff2e8r4en28t2ia2l8 0
charging foBirnd inrge Egneirogyn, esV containin g C-O groups. TBinhdieng Efnreargcy, teiVon of such carbon atoms iBsin di0ng. 4En7er.g yA, eV similar
vmaeluaseu roFFefidi gg0 uua.4rtre 8eU 77wb.. F=Fai ist+tt t7iionn Vbggt tatihthi neeise CC d0 1 .17sfso7 pp.r h hTotohhttoieose e lislesepc cdettrcruootenrnu t ssmopp ec ecomcttnrreadaa ousofucf AtrAievggdi tb bylala aticn ckUkd mumb ce=eeaa dss0u u wrrVeeid,td ha wa ttthhU Ueebr bs e==tar −−sa 7y7f, ,o0e0r la eantcnhtddre o7 7n sVVps.,.e wcthruicmh
make conducting regions that are not in direct contact with silver nanoparticles. Figure 7 shows
Table2.Bindingenergies(E ),peakwidths(W)andrelativeintensitiesofthepeaksdeconvolutedin
b
fitting the corresponding C 1s spectra, and Table 2 contains corresponding data.
theC1sandO1sspectraofAgblack.
It should be noted that the fitting the C 1s spectra with some components measured at Ub = −7
and 0 V, presented in Figure 7 is largely conditional and do not reflect the real relative
C1s O1s
concentrations of COx groups. At the same time, applying a positive bias voltage Ub = +7 V
Ub C-C/ C-OH/ C=O/ C-OH/
practically compensates the surface Cc=hOargiCn(gO, )Oand AFgig-Oure 7 (+7 V) reflects theC (Ore*a)Ol reCla(Otiv)Oe *
C-H C-O-C C(O*)O C-O-C
concentrations of COx groups.
E 284.7 286.2 287.6 289.8 530.6 532.5 533.5 534.7
b
0V W 1.45 1.50 1.51 1.58 1.42 1.65 1.80 1.85
6 Irel 0.48 0.35 60.10 0.07 0.18 0.21 5 +70 V.35 0.26
-7E Vb 284.6 286.2 2587.40 V 289.8 530.9 533.2 4 534.9 536.7
−7Intensity, a.u.V24 IEWrebl 2018..444.788 2018..15670.3 Intensity, a.u.2234018..727.367 2038..810.358 5013..027.467 5013..229.704Intensity, a.u. 23 5013..239.805 503.10.08 5013..319.008
+7V W 1.43 1.43 11.43 1.43 1.42 1.42 1 1.55 1.42 1.55
0 Irel 0.77 0.13 00.03 0.07 0.18 0.09 0 0.37 0.18 0.18
294 292 290 288 286 284 282 280 294 292 290 288 286 284 282 280 294 292 290 288 286 284 282 280
Binding Energy, eV Binding Energy, eV Binding Energy, eV

Figure 7. Fitting the C 1s photoelectron spectra of Ag black measured at Ub = −7, 0 and 7 V.
Antibiotics 2018, 7, x 9 of 19
Table 2. Binding energies (Eb), peak widths (W) and relative intensities of the peaks deconvoluted in
the C 1s and O 1s spectra of Ag black.
C 1s O 1s
Ub Heading C-C/ C-OH/ C=O/ C-OH/
C=O C(O)O Ag-O C(O*)O C(O)O*
C-H C-O-C C(O*)O C-O-C
Eb 284.7 286.2 287.6 289.8 530.6 532.5 533.5 534.7
0 V W 1.45 1.50 1.51 1.58 1.42 1.65 1.80 1.85
Irel 0.48 0.35 0.10 0.07 0.18 0.21 0.35 0.26
Eb 284.6 286.2 287.4 289.8 530.9 533.2 534.9 536.7
−7 V W 1.48 1.50 1.76 3.05 1.76 1.90 1.90 1.90
Antibiotics2018,7,80 8of18
Irel 0.47 0.17 0.23 0.13 0.24 0.27 0.38 0.00 0.10
Eb 284.8 286.3 287.7 288.8 530.7 532.4 532.5 531.8 533.8
+It7 sVh ouldWbe noted1.4th3 atth1e.4fi3t ting1t.4h3e C11s.4s3p ectra1.w42i thso1m.4e2 comp1o.5n5e ntsm1e.4a2s ureda1t.5U5 =−7
b
and0V,presenIrtele dinF0i.g7u7 re70is.1l3a rgel0y.0c3o ndit0i.o0n7 alan0d.1d8 onot0r.0e9fl ectth0e.3r7e alrela0t.1iv8e conc0e.n1t8r ations
ofCO groups. Atthesametime,applyingapositivebiasvoltageU =+7Vpracticallycompensates
x b
thesuItr fwacaes cfohuarngdin tgh,aat ntdheF pigeuarkes 7in(+ t7heV )lorwefl-eenctesrtghye rreegailorne loaft ivtheec Oon 1ces nstpraectitoran,s mofeCasOuregdr oaut pUs.b = −7
x
and 0I tVw (aFsigfouuren d8)t, hhaatvteh eclpoesea kbsininditnhge elonwer-geineesr agnydr eingtieonnsoitfietsh.e BOas1esds opne ctthrea ,rmefeeraesnucree ddaattaU [21=,2−2]7,
b
the binding energy of these peaks of about 530.8 eV cannot be attributed to C-O bonds. Therefore,
and0V(Figure8),haveclosebindingenergiesandintensities. Basedonthereferencedata[21,22],
tthheeyb isnhdoiunlgde bnee ragtytroibfuthteedse tpo etahkes Aofga-Obo buotn5d30s..8 AenVdc afrnonmot tbhee awttreiabku tdeedpteonCd-eOncbeo onfd Es.b Tahnedr eIfroelr oe,nt hUeby,
oshnoeu cldanb eaassttirginb ubteinddtiontgh eenAegr-gOiebs oonfd s5.3A0.n6 dafnrdom 53th0.e9w eeVa ktod eApegn-Adegn-Oce ostfaEte. aTnhdeI Irelo nofU th,ios nsetactaen,
b rel b
dasestiegrnmbinineddi nfrgoemn ethrgei efisttoifn5g3 t0h.6e aOn d1s5 s3p0e.9cterVa mtoeAasgu-Aregd- Oats Utabt e=. −T7h,e 0I andof 7t hVis, astraet e0,.2d4e,t 0e.r1m8 iannedd f0r.o1m8,
rel
rthesepfietcttiinvgeltyh.e O1sspectrameasuredatU =−7,0and7V,are0.24,0.18and0.18,respectively.
b
IItt sshhoouulldd bbee ssttrreesssseedd ththaat trerelalatitvivee inintetnensistiiteise sofo CfOCO grgoruopusp isn itnheth Oe O1s 1spsescpteructmru mmemaseuarseudr eadt Uabt
=U 7 V= 7coVrrceosrproenspdo tnhdosteh oosbetaoibnteadin ferdomfr othme trheelarteivlaet iCv e1Cs s1psescptreucmtru. Imt .mIteamnesa tnhsatt hthate tbhieasb ivaoslvtaogleta ogfe Uobf
b
= 7 V neutralize the surface charging.
U =7Vneutralizethesurfacecharging.
b
8
7 -7 V 8 0 V 5 +7 V
Intensity, a.u. 23456 Intensity, a.u. 246 Intensity, a.u. 1234
1
0 0 0
540 538 536 534 532 530 528 540 538 536 534 532 530 528 540 538 536 534 532 530 528
Binding Energy, eV Binding Energy, eV Binding Energy, eV

Figure8.FittingtheO1sphotoelectronspectrumofAgblackmeasuredatU =−7,0and7V.
b
Figure 8. Fitting the O 1s photoelectron spectrum of Ag black measured at Ub = −7, 0 and 7 V.
Figure9showsthesurveyspectrumoftheAg/bandagesystem.Alongwiththepeakscharacteristic
Antibiotics 2018, 7, x 10 of 19
ofsilFviegr,ucraer b9o nshaonwdso xtyhgee nsuthrevreeya rseppecetarkusmo foimf pthuer itAygsi/lbicaonndaatgoem ssy.stem. Along with the peaks
characteristic of silver, carbon and oxygen there are peaks of impurity silicon atoms.
6000
C 1s
u. O 1s
a.
sity, 4000
n
e
nt Ag 3d
I
O KVV
2000 C KVV
Si 2s
Si 2p
0
1200 800 400 0
Binding Energy, eV

Figure9.SurveyspectrumoftheAg/bandagesystem.
Figure 9. Survey spectrum of the Ag/bandage system.
In case of the Ag/bandage system the charge referencing was done using the C 1s spectrum
In case of the Ag/bandage system the charge referencing was done using the C 1s spectrum of
ofcellulose. Thelatterwassimulatedusingthereferencedata[24]byconsideringthedifferencein
cellulose. The latter was simulated using the reference data [24] by considering the difference in
peakresolution. Thebindingenergyof286.7eVwasassignedtoC-OHstateofcellulose. Figure10
peak resolution. The binding energy of 286.7 eV was assigned to C-OH state of cellulose. Figure 10
sshhoowwss tthhee CC 11ss ssppeeccttrruumm ooff tthheeA Agg//bbaannddaaggee ssaammppllee ffiitttteedd wwiitthh ffoouurr GGaauussssiiaann ppeeaakkss aatt 228866..77,, 228888..11,,
228844..88 aanndd 228833..1133 eeVV.. TThhee fifirrsstta annddt thhees seeccoonndd ppeeaakkss aarreea assssiiggnneedd ttoo cceelllluulloossee [[2244]].. TThhee tthhiirrdd ppeeaakk iiss
aassssiiggnneedd ttoo aaddvveennttiittiioouuss ccaarrbboonn,, wwhhiillee tthhee oorriiggiinn ooff tthhee ppeeaakk aatt 228833..11 eeVV iiss nnoott cclleeaarr bbeeccaauussee iitt ddooeess
nnoott ccoorrrreessppoonndd ttoo rreeffeerreennccee ddaattaa ffoorr ppoollyymmeerrss [[2244]].. HHoowweevveerr,, iitt mmaayy bbee rreessuulltteedd eeiitthheerr ooff ddiiffffeerreennttiiaall
charging or low-molecular weight species. Similar С 1s spectrum was recorded for Au/bandage
system. It slightly differs in relative concentration of C-C/C-H peak and peak at 283.1 eV. The O 1s
spectra of Ag/bandage and Au/bandage systems are practically indistinguishable. It means that the
peak at 283.1 eV may be assigned to C-C/C-H state as well.

283.13 ?
Au/bandage
Ag/bandage 3 283.12 (1.64) 3434
C-C/C-H
2 284.80 (1.60) 1693
u.
a. u.
ntensity, 1 cellulose ensity, a. 2
I C-OH Int 1
O-C-O
0 0
292 290 288 286 284 282 280 292 290 288 286 284 282 280
Binding Energy, eV Binding Energy, eV

Figure 10. The C 1s photoelectron spectra of Ag/bandage and Au/bandage systems.
Figure 11 shows the C 1s spectra of Ag black and Ag/bandage system. It is clearly seen that the
spectra are strongly different. The C 1s peak of the Ag/bandage system shifted to high binding
energy region by 0.66 eV and its FWHM is 1 eV more than that of Ag black. These differences may be
assigned to the size effect in photoelectron spectra which induces both energy shift to high binding
energy and the peak broadening [25,26]. The transition from AgNPs in the Ag black, to their
dispersion in the bandage followed with fairy large changes in size of AgNPs, and an increase of the
proportion of the Ag0 state was observed. Apparently, there was a partial reduction of silver and its
stabilization by a modified layer of cellulose. However, as follows from a comparison of the O 1s
Antibiotics 2018, 7, x 10 of 19
6000
C 1s
u. O 1s
a.
sity, 4000
n
e
nt Ag 3d
I
O KVV
2000 C KVV
Si 2s
Si 2p
0
1200 800 400 0
Binding Energy, eV

Figure 9. Survey spectrum of the Ag/bandage system.
In case of the Ag/bandage system the charge referencing was done using the C 1s spectrum of
cellulose. The latter was simulated using the reference data [24] by considering the difference in
peak resolution. The binding energy of 286.7 eV was assigned to C-OH state of cellulose. Figure 10
shows the C 1s spectrum of the Ag/bandage sample fitted with four Gaussian peaks at 286.7, 288.1,
284.8 and 283.13 eV. The first and the second peaks are assigned to cellulose [24]. The third peak is
aAsnstiigbinoteicds 2t0o1 8a,d7,v8e0ntitious carbon, while the origin of the peak at 283.1 eV is not clear because it 9doofe1s8
not correspond to reference data for polymers [24]. However, it may be resulted either of differential
charging or low-molecular weight species. Similar С 1s spectrum was recorded for Au/bandage
charging or low-molecular weight species. Similar C 1s spectrum was recorded for Au/bandage
system. It slightly differs in relative concentration of C-C/C-H peak and peak at 283.1 eV. The O 1s
system. ItslightlydiffersinrelativeconcentrationofC-C/C-Hpeakandpeakat283.1eV.TheO1s
spectra of Ag/bandage and Au/bandage systems are practically indistinguishable. It means that the
spectraofAg/bandageandAu/bandagesystemsarepracticallyindistinguishable. Itmeansthatthe
peak at 283.1 eV may be assigned to C-C/C-H state as well.
peakat283.1eVmaybeassignedtoC-C/C-Hstateaswell.

283.13 ?
Au/bandage
Ag/bandage 3 283.12 (1.64) 3434
C-C/C-H
2 284.80 (1.60) 1693
u.
a. u.
ntensity, 1 cellulose ensity, a. 2
I C-OH Int 1
O-C-O
0 0
292 290 288 286 284 282 280 292 290 288 286 284 282 280
Binding Energy, eV Binding Energy, eV

Figure10.TheC1sphotoelectronspectraofAg/bandageandAu/bandagesystems.
Figure 10. The C 1s photoelectron spectra of Ag/bandage and Au/bandage systems.
FFiigguurree 1111 sshhoowwss tthhee CC 11ss ssppeeccttrraa ooff AAgg bbllaacckk aanndd AAgg//bbaannddaaggee ssyysstteemm.. IItt iiss cclleeaarrllyy sseeeenn tthhaatt tthhee
ssppeeccttrraa aarree ssttrroonngglylyd dififfefreernetn.tT. hTehCe 1Cs p1se apkeoafkt hoef Athge/ bAagn/dbaagnedsaygset esmyssthemift esdhtioftehdig htob hinigdhin gbienndeirnggy
ernegeriogny breyg0i.o6n6 beVy 0a.n6d6 eitVs FaWndH iMts FisW1HeVMm iso 1re etVh amnotrhea tthoafnA tghabtl aocfk A.gT hbelaseckd.i Tffhereesnec deisffmeraeynbceesa mssaigyn beed
atosstihgenesdiz etoe ftfheec tsiinzep ehfofetocte lienc tprhoontsopeelecctrtraown hsipcehcitnrad uwcheiscbho itnhdeunceersg byosthhi fetnteorhgiyg hshbifint dtoin hgigehn ebrignydainngd
etnheerpgeya kanbdr otahdee npienagk [2b5r,o2a6d].enTihnegt r[a2n5s,2it6i]o. nTfhroe mtraAngsNitiPosn infrtohme AAggNblPacsk i,nt oththee iArgd ibsplaecrks,i otno inthtehire
dbiasnpdearsgieonfo ilnlo twhee dbawnidthagfea ifroyllloawrgeedc hwainthg efasiirny sliazregeo fcAhagnNgPess ,inan sdizaen oifn AcrgeNasPeso, afnthde apnr oinpcorretaiosen ooff tthhee
Antibiotics 2018, 7, x 11 of 19
pArgo0postratitoenw oafs thoeb sAergv0 esdta.teA wppaas roebnstelyr,vtehde.r Aepwpaasreanptlayr,t itahlerreed wuactsi oan poarftsiaillv reerdauncdtioitns osft asbilivliezra atinodn ibtsy
ssatpamebciotlridzaia fioteifo dtnhl aeb yyAe gra obmflaoccedklil fuaielnodds elAa.ygHe/bro awonfed vcaeeglrle,u aslsoyssfeote.l lmHow o(Fwsigfervuoermre, 1aa2sc) ,of otmhllepo awlorisws of-renonmoefr gtahy ce soOimdep1 saorfsi psthoeecnt lroaaft totehfre tmh Oeig A1hsgt
bbela acsksiagnndedA tgo/ tbhaen Adagg-Aegsy-Ost egmro(uFpig. u re12),thelow-energysideofthelattermightbeassignedtothe
Ag-Ag-Ogroup.
368.83
3
368.17
AgO
2 2
u.
a.
y, 2
sit
n 1
e
nt
I
1
0
378 376 374 372 370 368 366 364
Binding Energy, eV

Figure11.TheC1sphotoelectronspectraofAgblack(1)andAg/bandagesystem(2).
Figure 11. The C 1s photoelectron spectra of Ag black (1) and Ag/bandage system (2).
2 530.68
u.
a.
sity, 1
n
e
nt 1
I
2
3
0
538 536 534 532 530 528 526
Binding Energy, eV

Figure 12. The O 1s photoelectron spectra of Ag black (1), Ag/bandage (2), and Au/bandage systems (3).
But, given the almost complete coincidence of the O 1s spectra of Ag/bandage and Au/bandage
systems, this conclusion should be rejected. Thus, one can conclude that the basic state of Ag atoms
in Ag/bandage system is Ag0 state, whereas the oxidized silver is in the form of Ag-Ag-O groups,
and, as follows from Figure 12 the proportion of oxidized state is small. This is in accordance with
EXAFS and TEM data which indicate that silver atoms are mainly in Ag0 state. It should be noted
that EXAFS is not a surface-sensitive method as XPS and electron diffraction may be recorded only
from the ordered regions.
Table 3 shows the number of colony-forming units (CFU) of the studied microbes along the
perimeter of the bandage at a distance equal to the diameter of one colony on both sides of the edge
Antibiotics 2018, 7, x 11 of 19
spectra of the Ag black and Ag/bandage system (Figure 12), the low-energy side of the latter might
be assigned to the Ag-Ag-O group.
368.83
3
368.17
AgO
2 2
u.
a.
y, 2
sit
n 1
e
nt
I
1
0
378 376 374 372 370 368 366 364
Binding Energy, eV

Antibiotics2018F,i7g,u80re 11. The C 1s photoelectron spectra of Ag black (1) and Ag/bandage system (2). 10of18
2 530.68
u.
a.
sity, 1
n
e
nt 1
I
2
3
0
538 536 534 532 530 528 526
Binding Energy, eV

Figure 12. The O 1s photoelectron spectra of Ag black (1), Ag/bandage (2), and Au/bandage
Figure 12. The O 1s photoelectron spectra of Ag black (1), Ag/bandage (2), and Au/bandage systems (3).
systems(3).
But, given the almost complete coincidence of the O 1s spectra of Ag/bandage and Au/bandage
But,giventhealmostcompletecoincidenceoftheO1sspectraofAg/bandageandAu/bandage
systems, this conclusion should be rejected. Thus, one can conclude that the basic state of Ag atoms
systems,thisconclusionshouldberejected. Thus,onecanconcludethatthebasicstateofAgatoms
in Ag/bandage system is Ag0 state, whereas the oxidized silver is in the form of Ag-Ag-O groups,
inAg/bandagesystemisAg0 state,whereastheoxidizedsilverisintheformofAg-Ag-Ogroups,
and, as follows from Figure 12 the proportion of oxidized state is small. This is in accordance with
and,asfollowsfromFigure12theproportionofoxidizedstateissmall. Thisisinaccordancewith
EXAFS and TEM data which indicate that silver atoms are mainly in Ag0 state. It should be noted
EXAFSandTEMdatawhichindicatethatsilveratomsaremainlyinAg0state. Itshouldbenotedthat
that EXAFS is not a surface-sensitive method as XPS and electron diffraction may be recorded only
EXAFSisnotasurface-sensitivemethodasXPSandelectrondiffractionmayberecordedonlyfrom
from the ordered regions.
theorderedregions.
Table 3 shows the number of colony-forming units (CFU) of the studied microbes along the
Table 3 shows the number of colony-forming units (CFU) of the studied microbes along the
perimeter of the bandage at a distance equal to the diameter of one colony on both sides of the edge
perimeterofthebandageatadistanceequaltothediameterofonecolonyonbothsidesoftheedge
intheformMe(Q ;Q ),togetherwiththelevelofstatisticalsignificance,whereMeisthemedian,
1 3
Q —lowerquartile,Q —upperquartile.
1 3
Table3.Thenumberofcolony-formingunits(CFU)ofthestudiedmicroorganismsalongtheedgeof
thebandageatadistancetobothsidesoftheedgeequaltothediameterofonecolony(Me(Q ;Q ))
1 3
andthelevelofstatisticalsignificance(p)betweencontrolgroupsandgauzewithAgNPs.
StrainofMicroorganism Control(NormalBandage) AgNPs-ContainingBandage p
Staphylococcusaureus 7.0(6.0;8.0) 0.0(0.0;1.0) <0.001
Staphylococcushaemolyticus 11.0(7.5;14.5) 7.5(6.0;8.0) 0.049
Pseudomonasaeruginosa 5.0(5.0;6.0) 2.0(1.0;3.0) <0.001
Klebsiellapneumoniae 6.5(6.0;7.0) 2.0(1.0;3.0) <0.001
Escherichiacoli 16.0(13.5;17.5) 6.0(4.0;7.0) <0.001
Moraxellaspp. 8.0(5.0;8.5) 3.0(2.5;4.0) 0.002
Duetothefactthatthedataofthecontrolgroupsofdifferentstrainsdiffer,inordertocompare
theantibacterialeffectoftheAgNPs-containingbandagewithrespecttodifferentmicroorganisms,we
calculatedthepercentagereductionfactor. Table4presentstheresultsofthestudyofthepercentage
reductionofCFU.
Theresultsofthechangeintheantibacterialpropertiesoftheordinarymedicalgauzebandage
undertheinfluenceoflaserirradiationarepresentedinTable5,intheformMe(Q ;Q ),whereMeis
1 3
themedian,Q isthelowerquartile,Q istheupperquartile.
1 3