Graphene Oxide-Based Nanocomposites Decorated with Silver Nanoparticles as an Antibacterial ...

17 Pages·2017·3.51 MB·English

by  Sławomir Jaworski

Jaworskietal.NanoscaleResearchLetters (2018) 13:116
https://doi.org/10.1186/s11671-018-2533-2
NANO EXPRESS Open Access
Graphene Oxide-Based Nanocomposites
Decorated with Silver Nanoparticles as an
Antibacterial Agent
Sławomir Jaworski1, Mateusz Wierzbicki1, Ewa Sawosz1, Anna Jung2, Grzegorz Gielerak2, Joanna Biernat3,4,
Henryk Jaremek3, Witold Łojkowski5, Bartosz Woźniak5, Jacek Wojnarowicz5, Leszek Stobiński6, Artur Małolepszy6,
Marta Mazurkiewicz-Pawlicka6, Maciej Łojkowski7, Natalia Kurantowicz1 and André Chwalibog8*
Abstract
One ofthe most promising methods against drug-resistant bacteria can be surface-modified materials withbiocidal
nanoparticles and nanocomposites. Herein, we present a nanocompositewith silver nanoparticles(Ag-NPs) onthe
surfaceofgrapheneoxide(GO)asanovelmultifunctionalantibacterialandantifungalmaterial.Ultrasonictechnologies
havebeenusedasaneffectivemethodofcoatingpolyurethanefoils.Toxicityongram-negativebacteria(Escherichia
coli),gram-positivebacteria(StaphylococcusaureusandStaphylococcusepidermidis),andpathogenicyeast(Candida
albicans)wasevaluatedbyanalysisofcellmorphology,assessmentofcellviabilityusingthePrestoBlueassay,analysis
ofcellmembraneintegrityusingthelactatedehydrogenaseassay,andreactiveoxygenspeciesproduction.Compared
toAg-NPsandGO,whichhavebeenwidelyusedasantibacterialagents,ournanocompositeshowsmuchhigher
antimicrobialefficiencytowardbacteriaandyeastcells.
Keywords:Grapheneoxide,Silvernanoparticles,Antimicrobialproperties
Background relatedbacteria,whicharethenabletopassontheresist-
The development of antibiotics has played a significant ance genes to subsequent generations. Thus, the emer-
role in controlling the number of bacterial infections. gence of antibiotic-resistant bacteria represents a serious
However, the improper use and the overuse of antibiotics problem that could be overcome by the development of
have led to the development of multidrug resistance in novel antimicrobial agents. Antibacterial agents are very
many bacterial species. Some strains have become importantinthetextileindustry,waterdisinfection,medi-
resistant to practically all of the commonly available cine, and food packaging. Nanoparticles and nanomater-
agents: beta-lactams, tetracyclines, and aminoglycosides ials can be used as an alternative to antibiotics [4]. The
[1]. The major resistant pathogens are methicillin- mechanismofantibacterial activity of nanoparticlesvaries
resistant Staphylococcus aureus, vancomycin-resistant among the different types of nanoparticle. While some
Enterococcus, and extended-spectrum β-lactamase- proposed mechanisms relate to the physiochemical struc-
producing Klebsiella pneumoniae and Escherichia coli [2, tureofthenanoparticles,othersrelatetotheincreasedre-
3].Bacteria,withtheirverylargepopulationsandfastpro- lease of antibacterial ions from nanoparticle surfaces.
liferation time, are able to rapidly develop mechanisms of Multiple simultaneous mechanisms of action against mi-
antibioticresistancewhenasubsetofthebacteriapopula- crobes would require a variety of synchronous DNA mu-
tion survives antibiotic treatment. Moreover, antibiotic- tations in the same microbial cell for the development of
resistant bacteria are able to transfer copies of DNA that resistance; therefore, it is difficult for bacterial cells to be-
code for a mechanism of resistance to other distantly come resistant to nanoparticles and nanomaterials. Anti-
microbial nanomaterials, such as silver, copper, fullerenes,
*Correspondence:[email protected] andsingle-walledcarbonnanotubes,mayofferseveralad-
8DepartmentofVeterinaryandAnimalSciences,UniversityofCopenhagen, vantages due to their unique physicochemical properties
Groennegaardsvej3,1870Frederiksberg,Denmark and high surface areas [5–8]. The exact mechanisms of
Fulllistofauthorinformationisavailableattheendofthearticle
©TheAuthor(s).2018OpenAccessThisarticleisdistributedunderthetermsoftheCreativeCommonsAttribution4.0
InternationalLicense(http://creativecommons.org/licenses/by/4.0/),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedyougiveappropriatecredittotheoriginalauthor(s)andthesource,providealinkto
theCreativeCommonslicense,andindicateifchangesweremade.
Jaworskietal.NanoscaleResearchLetters (2018) 13:116 Page2of17
nanoparticle (NP) toxicity against various bacteria are not GO sheets via covalent and non-covalent interactions.
completelyunderstood.Accordingtothecurrentresearch, The strong antibacterial activity of GO has been
the major processes underlyingthe antibacterialeffects of reported. The antibacterial activity of GO has been
NPs are disruption of the bacterial cell membrane, metal assigned to membrane stress induced by sharp edges of
ionrelease,generationofROS,penetrationofthebacterial graphene oxide nanosheets, which may result in physical
cellmembrane,andinductionofintracellularantibacterial damage to cell membranes, leading to the loss of
effects, including interactions with DNA and proteins [9, bacterial membrane integrity [21]. Recently, graphene-
10].NPsareabletoattachtothemembraneofbacteriaby functionalized antimicrobial nanoparticles have been
electrostatic interaction and disrupt the integrity of the used as promising antibacterial materials [22, 23].
bacterial membrane. The positive charge of the surface of Nanocomposites can overcome the limitations of the in-
the NPs is essential for the adhesion. The positive charge dividual components. For example, antibacterial nano-
enables electrostatic addition between NPs and negatively materials attached to the graphene substrate are more
charged cell membrane of the microorganisms [11]. The stable and well dispersed [24]. These nanocomposites
electrostatic connection between NPs with the sulfur- couldcontainmetals,metaloxides,andpolymers.
containing proteins present on the surface of bacterial One of the most promising methods against drug-
cells causes irreversible changes in cell wall structure resistant bacteria can be surface-modified materials with
resultingin damages ofcellwalland membrane[12].The biocidal nanoparticles. Ultrasonic technologies have
bacterial membrane is crucial, irrespective of the meta- been confirmed as an effective method of coating
bolicstatusofthecell,asitprovidesselectivepermeability various materials with antibacterial and fungicidal sub-
for cellular homeostasis and metabolic energy transduc- stances [25–28]. Many researchers classify the ultra-
tion. The second antibacterial and antifungal activity of sound method as a “green technology” [29, 30]. The
NPsisduetotheirabilitytoproduceROSandfreeradical method is based on the use of cavitation phenomena,
species [13]. Increased level of ROS induced hyperoxida- which is the formation, growth, and collapse of cavita-
tionoflipids,proteins,andDNA[14]. tion bubbles in the liquid medium [31, 32]. Imploding
Moreover, the structures of many types of NPs are bubbles generate immense amounts of energy in micro-
suitable for carrying antimicrobial agents [15, 16]. regions up to 5000 K and pressure up to 2000 atm
Carriers can help to protect the drugs from resistance by within a short period of time [33, 34]. Consequently,
target bacteria. A nanoparticle-based drug delivery sys- shock waves and so-called microjets directed toward the
tem can help to target antibiotics to an infection site solid surface are generated [35]. Located in a liquid
and thereby minimize systemic side effects. Other ad- medium, NPs are driven up by the implosion effect and
vantages include improved solubility of hydrophobic jetstreamsathighspeed(>100m/s)onthesolidsurface
drugs, prolonged systemic circulation time and drug and form a layer [36]. Acoustic cavitation can also lead
half-life,andsustaineddrugrelease[4]. to change in the physical properties of sonicated objects,
Recently, it has been demonstrated that graphene, a e.g.,resizingofGOflakes[37,38].
new allotrope of carbon, has antibacterial activity. We achieved promising results in our previous studies
Graphene is a material made of carbon atoms that are with Salmonella enterica and Listeria monocytogenes
bonded together in a repeating pattern of hexagons. A treated with pristine graphene, GO, and reduced GO
unique feature ofgraphene flakes istheratio ofitsthick- [20]. Of the different types of graphene, GO was also
ness to the surface. The surface of graphene is covered found to have the highest antibacterial activity at a low
with an electron cloud, which probably predisposes this concentration. Bacterial cells were distributed over the
material to be an electron donor and gives it the ability entire surface of the GO. In this study, we hypothesized
to make special bonds. The edges of graphene have that GO decorated with silver nanoparticles (GO-Ag)
other bonds (characteristic for diamond sp3 type bonds), will havestronger toxic influence on microbial cells than
and these places may have different physicochemical bare GO or silver nanoparticles (Ag-NPs). Because it has
characteristics [17]. These characteristics suggest that two active sides (surface and edges), GO oxide can
graphene can be exposed to plastic adhesion to different attach Ag-NPs to the edges and adhere to the cell sur-
intercellular structures, including bacterial cells [18–20]. face. The antibacterial activity of graphene-based nano-
In addition, because it has two active sides (surface and composites may be due to the disruption of the cell
edges), graphene can attach biological molecules to its membrane and oxidative stress. The objective of this
edges and adhere to the cell surface. An oxidized form study was to evaluate the antimicrobial activity of GO-
of graphene, graphene oxide (GO), is easily dispersible based nanocomposites decorated with Ag-NPs in com-
in water and other organic solvents due to the presence parison to bare GO and Ag-NPs using gram-negative
ofthe oxygen functionalities. Theoxygenated groups en- bacteria (Escherichia coli), gram-positive bacteria
able the straightforward chemical functionalization of (Staphylococcus aureus and Staphylococcus epidermidis),
Jaworskietal.NanoscaleResearchLetters (2018) 13:116 Page3of17
and pathogenic yeast (Candida albicans) using an in lefttodry.After itwasdried,thespectrum wascollected
vitro model. The investigation consisted of structural intherange400–4000cm−1.
analysis of nanocomposites using X-ray diffraction, Average particle size and zeta potential measurements
Raman spectroscopy transmission, FT-IR, electron mi- werecarriedoutusingZetasizerNano-ZSZEN3600pro-
croscopy (TEM), scanning electron microscopy (SEM) duced by Malvern Instruments Ltd. (Malvern, UK) using
and atomic force microscopy (AFM), evaluation of mi- the dynamic light scattering (DLS) mode and laser Dop-
crobial cell morphology, assessment of cell viability by pler electrophoresis, respectively, at room temperature
PrestoBlue™ assay, investigation of cell membrane integ- (23°C).
rity by lactate dehydrogenase assay (LDH), and assess-
mentofreactive oxygenspecies(ROS)production.
TEM/SEM/AFMAnalysisofNanomaterials
The morphology of powders and foils was determined
Methods
using the transmission electron microscope (TEM;
Synthesis,Modification,andCharacterizationofGraphene
JEM-1220 JEOL, Tokyo, Japan, accelerating voltage of
Oxide
80 kV) and scanning electron microscope (SEM; Zeiss,
In this study, a commercially available graphite powder
Ultra Plus, Oberkochen, Germany). Samples for TEM
(Acros Organics, New Jersey, USA) was oxidized by the
observations were prepared by placing droplets of hydro-
modified Hummers method [39]. Ten grams of graphite
colloidsontoTEMgrids(Formvaron3mm200MeshCu
powder were mixed with 230 mL of concentrated sul-
Grids, Agar Scientific, Stansted, UK). Immediately after
furic acid (98%) below 10 °C. Then, 4.7 g of sodium ni-
air-drying the droplets, the grids were inserted into the
trate and 30 g of potassium permanganate were added
microscope.
gradually to the sulfuric acid and graphite mixture while
For SEM analysis, samples were coated with a thin
maintaining the temperature below 10 °C. Then, the
carbon layer using the sputter coater (SCD 005/CEA
mixture was heated to 30 °C and stirred for 2 h. In the
035, BAL-TEC, Pfäffikon, Switzerland). An internal la-
next step, 100 mL of water was added, and the mixture
boratory measurement procedure was applied (P5.10,
temperature reached ~100 °C. Finally, the mixture was
edition 6of26.08.2015).
treated with 10 mL of hydrogen peroxide. For purifica-
AFM (atomic force microscopy) imaging was carried
tion, the slurry was filtrated and washed with deionized
out using Asylum Research MFP3D Bio software
water untilthepHofthefiltrate reached6.5.
(version: Asylum Research MFP3D 15.106.09). Surface
X-ray diffraction patterns of GO were gathered at
topography imaging and detection of GO on the tested
roomtemperaturewithintherangeof2thetaanglefrom
foil surfaces were carried out using two imaging modes,
10° to 100° with the step of 0.02° using the X-ray powder
AC mode for phase contrast imaging and lateral force
diffractometer (CuKα1) (X’Pert PRO, PANalytical, microscopy (LFM) for GO detection since GO reduces
Almelo,Netherlands).
friction forces[40].
The analysis of carbon, hydrogen, nitrogen, and sulfur
content by weight in GO was carried out using theVario
ELIIIapparatusproducedbyElementarAnalysensysteme PreparationofPolyurethaneFoilsCoatedwithGOand
GmbH (Langenselbold, Germany). Prior to performing Ag-NPs
measurements of chemical analyses of GO, the samples For covering polyurethane foils, suspensions of Ag-NPs
were subject to 24-h desorption in a desorption station (HydroSilver1000, Amepox, Łódź, Poland) and GO were
(VcPrep 061, Micromeritics, Norcross, GA, USA) under used. Suspensions of GO, Ag-NPs, and GO-Ag (GO
vacuum (0.05 mbar) at 50 °C. Oxygen content was calcu- (200 μg/mL), Ag-NPs (100 μg/mL), GO (200 μg/mL)+
lated by subtracting the determined contents of carbon, Ag-NPs (100 μg/mL)) were prepared in deionized water
hydrogen,nitrogen,andsulfurfrom100%weight. (conductance 0.09 μS/cm, deionizer: HLP 20UV, Hydro-
Raman spectroscopy was performed using an inVia lab, Staszyn, Poland). The suspensions were used with-
Ramanmicroscope (Renishaw, UK). Graphene oxide was outadditionalpurification andfiltration.
analyzed with the 514-nm laser wavelength with the 5% Ultrasonic coating of polyurethane foils (15×15×0.
of its initial power. The spectra were collected from five 05 mm) took place in a glass flask with a volume of
different spots on the sample. The exposure time was 50 ml. Foil samples were fastened on a stand (Teflon)
10sandtwoscanswere collected. andsubsequently immersedinthepreparedsuspensions.
FT-IR measurements were performed using Nicolet The coating process was performed using an ultrasonic
iS10 spectrometer(ThermoFisher Scientific,USA)inat- horn (Ti horn, Ø13 mm, 60% efficiency, 20 kHz, Sonics
tenuated total reflectance mode on a diamond crystal. & Materials, Inc., Newtown, CT, USA) placed square to
Five microliters of graphene oxide water suspension was the foil samples present in the suspension. The process
dripped on the surface of the diamond crystal and it was temperature was 30±1 °C. The covered samples were
Jaworskietal.NanoscaleResearchLetters (2018) 13:116 Page4of17
flushed in deionized water and dried in a laminar cham- without nanoparticles were used as control group. The
ber andsubsequentlypacked insterilepackages. bacteria and yeast growth under the foils was measured
after24hofincubationat37°C.
SurfaceFreeEnergy
Wettability tests were carried out using the Data Physics
ViabilityAssay
OCA– 20goniometer(DataPhysics InstrumentsGmbH,
Cell viability was evaluated using the PrestoBlue™ Cell
Filderstadt,Germany).Surfacefree energy(SFE) was cal-
Viability Assay (Life Technologies, Taastrup, Denmark).
culated using the Owens, Wendt, Rabel, and Kaelble
PrestoBlue™ reagent is quickly reduced by metabolically
(OWRK) method using two test liquids: deionized water
active cells, providing a quantitative measure of viability
anddiiodomethane [41].
and cytotoxicity. Bacterial and yeast cells were cultured
onto foils coated with GO, Ag-NPs, and GO-Ag located
BacterialandYeastCultivationandPreparation
on inserts inserted into 6-well plates (200 μL MH broth
Staphylococcus aureus (ATCC 25923) and Staphylococ-
with 5×103 cells per foil) and incubated for 24 h. In the
cus epidermidis (ATCC 14990), Escherichia coli (ATCC
next step, 90 μL of each sample was transferred to 96-
25922), and Candida albicans (90028) were obtained
well plates and 10 μL of PrestoBlue™ reagent was added
from LGC Standards (Lomianki, Poland). The strains
to each well and incubated for an additional 2 h at 37 °
were stored as spore suspensions in 20% (v/v) glycerol at
C. The optical density of each well was recorded at
−20 °C. Prior to their use in experiments, the strains
570 nm on an enzyme-linked immunosorbent assay
were defrostedandthe glycerolwasremovedbywashing
(ELISA) reader (Infinite M200, Tecan, Durham, NC,
the bacterial cells with distilled water. The bacteria and
USA). Cell viability was expressed as the percentage
yeast were then grown on the following nutrient media:
(OD −OD )/(OD −OD )×100%, where
tryptic soy agarfor S.aureus andE.coli,brain heart agar test blank control blank
OD is the optical density of cells exposed to tested
for S. epidermidis, and Sabouraud’s agar for C. albicans test
foils, OD is the optical density of the control sam-
(Merck Millipore, Darmstadt, Germany). The bacteria control
ple, and OD is the optical density of wells without
and yeast grown on agar plates were harvested by gently blank
bacterialandyeastcells.
washing the plates with sterile distilled saline solution.
To calculate the number of bacteria in the cell suspen-
sion, the optical density of the suspensions at 600 nm MembraneIntegrity
(OD ) was measured using a spectrophotometer An LDH test (In Vitro Toxicology Assay Kit, lactic de-
600
(Helios Epsilon, Unicam, Milwaukee, WI, USA). Calibra- hydrogenase based, Sigma-Aldrich, Hamburg, Germany)
tion curves for each of the microorganisms were pre- was used to evaluate cell membrane integrity. The
pared by performing serial tenfold dilutions (up to 10−5) resulting reduced NAD (NADH+) was utilized in the
of bacterial and yeast suspensions of known optical stoichiometric conversion of a tetrazolium dye. When
density. One milliliter of each dilution was spread on cell-free aliquots of the medium from cultures were
petri dishes containing the nutrient medium. After 24 h assayed, the amount of LDH activity could be used as an
of incubation at 37 °C, the number of colonies formed indicator of membrane integrity. If the membrane was
onthepetridishes wasenumerated.Based ontheresults damaged, intracellular LDH molecules were released
of the enumerations (conducted in triplicate), the into the culture medium. Bacterial and yeast cells were
density of the original bacterial suspension in colony- cultured on foils (GO, Ag-NPs, and GO-Ag) located on
formingunits(CFU)/mLwascalculated. inserts inserted in 6-well plates (200 μL MH broth with
5×103 cells per foil) and incubated for 24 h. Cells
AntimicrobialAssay cultured on foil without nanoparticles were used as a
The inoculum for the antibacterial assay was prepared control. After this time, the samples were transferred to
from actively growing organisms (logarithmic phase). microcentrifuge tubes and centrifuged at 1200 rpm for
The inoculums of all microorganisms were prepared 5 min. One hundred microliters of supernatant were
from an overnight culture grown aerobically in Mueller– transferred to 96-well plates, and 100 μL of the LDH
Hinton (MH) broth at 37 °C. The bacterial and yeast assay mixture was added to each well. The plate was
concentration was determined by measuring optical covered and incubated for 30 min at room temperature.
density at 600 nm (OD ). Briefly, bacterial and yeast The optical density of each well was recorded at 450 nm
600
suspensions were prepared from overnight cultures and on an ELISA reader (Infinite M200, Tecan, Männedorf,
adjusted to 106 CFU/ml. Inoculum was inoculated Switzerland). LDH leakagewas expressedasthe percent-
evenly onto the surface of MH agar in petri dishes by age {(OD −OD )−(OD −OD )/(OD
test blank control blank control
swabbing. Sterile foils coated with GO, Ag-NPs, and −OD )}×100%, where OD is the optical density of
blank test
GO-Ag were deposited onto the agar surface. Foils cells exposed to tested foils, OD is the optical
control
Jaworskietal.NanoscaleResearchLetters (2018) 13:116 Page5of17
density of the control sample, and OD is the optical ofgraphite,sodium nitrate,andprobablyareducedform
blank
density ofwellswithoutcells. ofgrapheneoxide.
Raman spectroscopy can give information about the
structural features of graphene oxide. The D band is at-
SEMAnalysisofMicroorganisms
tributed to the structural disorder, while the G band
Prior to SEM analysis, samples of bacteria and yeast in-
comes from the bond stretching of carbon sp2 atoms
cubated on foils with GO-Ag and untreated bacteria
[42]. The additional bands (including D′, 2D, and D+G)
were prepared. Briefly, a drop of bacterial and yeast cul-
ture (106 CFU/ml) was incubated on foils with GO-Ag arise from the defects present in the graphitic structure
of the carbon material. I /I ratio (calculated from the
nanocomposite, or untreated bacteria was deposited on D G
intensity of D and G bands) can be used to characterize
the surface of a sterile cover glass and incubated for
the disorder of the graphitic structure in carbon
24 h at 37 °C inside an empty petri dish. All samples
materials. As can be seen in Fig. 2, GO has a highly
were dried and covered with gold. Finally, the samples
disordered structure due to many functional groups in
were imaged with SEM (FEI Quanta 200, Tokyo, Japan)
the structure formed during oxidation of graphite
atanaccelerationvoltageof15kV.
powder. The position of the D band is 1351 cm−1 and
theGband1590cm−1;theI /I ratiois1.15.
D G
ROSProduction
The FT-IR spectrum of graphene oxide collected in the
ROS production was evaluated using DCFDA, Cellular
ATRmoderevealedthatGOhasalotoffunctionalgroups
Reactive Oxygen Species Detection Assay Kit (Abcam,
presentinthestructure.Themostnotablepeakcanbeob-
Cambridge, UK). DCFDA uses the cell permeant reagent served at ~3500 cm−1, which is assigned mainly to water
2′,7′–dichlorofluorescein diacetate, a fluorogenic dye
and hydroxyl groups (Fig. 3). Avery intensivepeakaround
that measures hydroxyl, peroxyl, and other ROS activ- 1080 cm−1 can also be attributed to hydroxyl groups. The
ities within the cell. After diffusion into the cell, DCFDA peak around 1600 cm−1 usually is assigned to C=C bonds
is deacetylated by cellular esterases to a non-fluorescent
present in graphitic carbon. However, our previous XPS
compound, which is later oxidized by ROS into 2′,7′–
studies show that there is a few C=C bonds in graphene
dichlorofluorescein (DCF). Bacterial and yeast cells were
oxide [43]; hence, we attribute this peak to mostly water
cultured on foils (GO, Ag-NPs, and GO-Ag) located on
still present in the graphene oxide. Other peaks observed
inserts inserted in 6-well plates (200 μL MH broth with
ontheFT-IRspectrumshowthatGOisrichingroupscon-
5×103 cells per foil) and incubated for 24 h. Cells
taining C=O bonds (mainly carboxyl groups), peak around
cultured on foil without nanoparticles were used as a 1720 cm−1, epoxy (C–O–C) with the visible peak around
control. After this time, the samples were transferred to 1200cm−1,andC–Hbonds(peakaround2800cm−1).The
microcentrifuge tubes and centrifuged at 1200 rpm for
FT-IR analysis is in good agreement with XPS measure-
5 min. One hundred microliters of supernatant were
ments performed for graphene oxide where also hydroxyl,
transferred to 96-well plates, and 100 μL of diluted
carboxyl, epoxy, and carbonyl groups were identified [44].
DCFDA was added to each well and incubated for an
GO and GO after a 10-min ultrasonic homogenization
additional 45 min at 37 °C in the dark. DCF production
were compared (Figs. 4 and 5), and similarly, Ag-NPs with
was measured by fluorescence spectroscopy with an ex-
Ag-NPs after a 10-min ultrasonic homogenization (Figs. 4
citation wavelength at 485 nm and an emission wave-
and 6) were compared. In order to avoid changes to com-
length at 535 nm on an ELISA reader (Infinite M200,
pound morphology, all compounds were rapidly cooled
Tecan,Durham,NC,USA).
down with liquid nitrogen and dried in a lyophilizer.
Figure 5a, b presentsGO flakes, whileFig. 5c,d shows the
Results effectsof ultrasounds on GO flakes, which undergo partial
CharacteristicsofGOandAg-NPs foldingandfragmentation.Figure6alsoshowsasimilaref-
The chemical analysis revealed the presence of nitrogen, fectforAg-NPs,whereachangeofmaterialmorphologyis
carbon, sulfur,hydrogen, andoxygen(Table1). visible.Figure6a,bdisplaysdriedpoly(vinylalcohol),which
The phase analysis of the GO sample (Fig. 1) revealed was used for stabilizing the water suspension of Ag-NPs.
the presence of impurities coming from trace quantities The destructive effect of ultrasonic homogenization was
notableasthepolyvinylalcoholstructurewasbrokendown
intolongheterogeneouspartswithsmallsphericalopenings
Table1Resultsofchemicalanalysesofgrapheneoxide
(Fig.6c,d).
samples
Sample Element(%wt)
AverageSizeandZetaPotential
N C S H O
The results of average particle/agglomerate size in water
Grapheneoxide 0.042 48.41 0.390 1.963 49.195
suspensions are presented in Table 2. Analyses of
Jaworskietal.NanoscaleResearchLetters (2018) 13:116 Page6of17
Fig.1X-RaydiffractionpatternsofGOpowders.ThephaseanalysisoftheGOsamplerevealedthepresenceofimpuritiescomingfromtrace
quantitiesofgraphite,sodiumnitrate,andprobablyareducedformofgrapheneoxide
average size were carried out for concentrated suspen- suspension stabilization. The large standard deviation of
sions which were not subject to ultrasonic the Ag-NP sample homogenized by ultrasound resulted
homogenization (as received) and for diluted suspen- from the presence of both loose Ag-NPs and Ag-NPs
sions. Diluted suspensions before the test were subject driven into the poly(vinyl alcohol) in the suspension. In
to ultrasonic homogenization, with homogenization the case of GO suspension, the average particle size of
parameters identical to those used during the ultrasonic the sample subject to ultrasonic homogenization was
coating of the foil with nanomaterial layers (Ag-NPs, 263 nm and was ca 7.7 times smaller than the average
GO). For Ag-NP suspension, the action of the particle size of the sample that was not subject to
ultrasounds caused an increase in the average particle homogenization. The obtained results are convergent
size from 80 to 218 nm. The main cause of the increase with the SEM tests (Fig. 5), which show the destructive
in the average particle size after ultrasonic effect of ultrasounds on GO flakes. The decrease of the
homogenization in the suspension (apart from the average GO particle size was caused by defragmentation
process of Ag-NP agglomeration) was that Ag-NPs were or folding of the GO flakes. However, it should be
driven into the poly(vinyl alcohol) that was used for emphasized that the results of the average particle size of
Fig.2RamanspectrumofgrapheneoxidewithproposeddeconvolutionoftheD,G,D′,2D,andD+Gbands.GOhasahighlydisorderedstructure
duetomanyfunctionalgroupsinthestructureformedduringoxidationofgraphitepowder.ThepositionoftheDbandis1351cm−1andtheGband
1590cm−1;theI /I ratiois1.15
D G
Jaworskietal.NanoscaleResearchLetters (2018) 13:116 Page7of17
Fig.3FT-IR(ATR,attenuatedtotalreflectance)spectrumofgrapheneoxidewithproposedassignmentoffunctionalgroupspresentinGO.The
mostnotablepeakswereobservedat~3500cm−1,(attributedtowaterandhydroxylgroups),~1080cm−1(hydroxylgroups),~1600cm−1(assignedto
C=Cbondspresentingraphiticcarbon).OtherpeaksobservedontheFT-IRspectrumshowthatGOisrichingroupscontainingC=O(mainlycarboxyl
groups),peakaround1720cm−1,epoxy(C–O–C)withthevisiblepeakaround1200cm−1,andC–Hbonds(peakaround2800cm−1)
GO suspension samples involve an error related to the sample of Ag-NP suspension was stabilized sterically by
nanomaterial shape. The results obtained by the DLS preserving Ag-NP distances through poly(vinyl alcohol)
method are a hydrodynamic average that is calculated addition, which prevented agglomeration/aggregation of
basedontheshapeofaspherethathasthesamediffusion Ag-NPs. The zeta potential of the GO suspension sam-
coefficient as the measured particles; however, the shape ple, in turn, was −41 mV, which gave a moderate elec-
ofGOwasflakes,whichwasconfirmedbySEMimages. trostatic stability to the sample. A moderate electrostatic
Test results of the zeta potential analysis of samples stability of a sample is characterized by slow sedimenta-
are provided in Table 3. The zeta potential of Ag-NPs in tion with virtually negligible change of particle size in
a water suspension was merely −5.9 mV, which resulted theperiodofdeclaredfitnessofthesuspension.Thezeta
in a lack of electrostatic stability of the sample. The potential result of the mixture of Ag-NPs and GO was
Fig.4TEMimagesofagglomeratedGOflakes(a),GOflakesafterultrasonictreatment(b),agglomeratedAg-NPs(c),Ag-NPsafterultrasonictreatment
(d),andGO-Ag(e,f).ThedecreaseoftheaverageGOparticlesizeafterultrasonictreatmentwascausedbydefragmentationorfoldingoftheGO
flakes.ThedecreaseoftheaverageAgsizeafterultrasonictreatmentwascausedbydefragmentationofAgagglomerates.Note:Arrowspoint
toAg-NPs/agglomerates
Jaworskietal.NanoscaleResearchLetters (2018) 13:116 Page8of17
Fig.5ComparisonofmorphologyoflyophilizedGOflakes(a,b)andGOflakesafterultrasonictreatment(c,d)usingscanningelectronmicroscopy.
ThedecreaseoftheaverageGOparticlesizeafterultrasonictreatmentwascausedbydefragmentationorfoldingoftheGOflakes
−7.1 mV, which potentially means that during the ac- FoilCharacteristics
tion of the ultrasounds, the GO flakes were coated by In order to determine the morphology of the created
poly(vinyl alcohol) and Ag-NPs. The obtained zeta layers, four types of foil samples were compared (Fig. 7):
potential result of the mixture of Ag-NPs and GO pure polyurethane foil (A, B), GO-coated polyurethane
sample in the water suspension implied that electro- foil (C, D), Ag-NP-coated polyurethane foil (E, F), and
static stability was not present. GO-Ag mixture-coated polyurethane foil(G,H).
Fig.6ComparisonofmorphologyoflyophilizedAg-NPmixture(a,b)andAg-NPmixtureafterultrasonictreatment(c,d)usingscanningelectronmicroscopy
Jaworskietal.NanoscaleResearchLetters (2018) 13:116 Page9of17
Table2Testresultsofaverageparticle/agglomeratesizein those in Fig. 8e, f. Figure 8c, d shows GO-Ag-NP-coated
suspensions film.Figure8eshowsthesurfaceofthefoilalmostentirely
Sample SizebyDLS,Z-average, coveredwithGOflakes;thephasecontrastimagehelpsto
diameter[nm]±SD observe these two phases, the darker area is GO and the
Ag-NPs(asreceived) 80±1 lighterareaispolymerfoil.Itwasnoticedthatthemorph-
Ag-NPs(100μg/mL,afterUS) 218±93 ology of the foils has changed after Ag-NP coating
comparing to the not-coated foils. The GO-Ag-NP
GO(asreceived) 2030±36
coating differs from the previous one because it
GO(200μg/mL,afterUS) 263±8
contains also small amounts of GO flakes seen as
GO(200μg/mL)-Ag(100μg/mL) 251±10
small black spots on the image, as it was mentioned
DLSdynamiclightscattering,Z-averageharmonicintensityaveragedparticle earlier. Figure 8f depicts magnification of one GO
diameter,SDstandarddeviation,USultrasoundmethod,Ag-NPs(asreceived)
HydroSilver1000(Amepox,Łódź,Poland)materialwasnotprocessedAg-NPs flake made in LFM. The reduced friction confirms
(100μg/mL,afterUS)asabovebutwasprocessedunderUSundersimilar that it is, in fact, a GO flake.
conditionstothefoilsintheprecedingsections,GO(asreceived)materialnot
processed,GO(200μg/mL,afterUS)asabovebutprocessedunderUSunder Thepolarcomponent for the GO-coated foil increased
similarconditionstothefoilsintheprecedingsections,GO(200μg/mL)-Ag in relation to pure foil, from 2.3±0.6 to 68.9±2.8 mJ/
(100μg/mL)materialsmixedtogetherandprocessedunderUSundersimilar m2, while the dispersion component decreased from 34.
conditionstothefoilsintheprecedingsections
4±1.3 to 8.2±1.2 mJ/m2. SFE increased from 36.7±1.4
Figure 7a, b shows an uncoated polyurethane foil with to 77.0±3.4 mJ/m2. A similar effect was not observed
a smoothsurface with singleimpurities.In Fig.4c, d, the on foil surfaces coated with Ag-NPs and GO-Ag mix-
broken-down GO flakes deposited on the polyurethane ture. SFE of foil samples coated with Ag-NPs and GO-
foilsurfacearenoticeable. Figure7e,fshowsthefoilsur- Agmixturedoesnot differstatistically(Table4).
face coated with Ag-NPs on which grid structures com-
posed of polyvinyl alcohol and Ag-NPs are observable. AntibacterialProperties
Figure 7g, h presents a mixture of GO-Ag composition, The antibacterial activity of the different foils coated
which was mixed under the influence of ultrasounds and with GO, Ag-NPs, and GO-Ag were tested with E. coli,
depositedonthefoilsurface. S. aureus, S. epidermidis, and C. albicans. Results
showed that after co-incubation with bacteria at 37 °C
AFMAnalysisandSurfaceFreeEnergy for 24 h, foils inhibited the growth of all tested microor-
AFM and LFM were used to complement the informa- ganisms but to various degrees. The maximum antibac-
tion about the surface morphology investigated by SEM. terial effect against all tested microorganisms was with
The investigation confirmed evolution of surface foil coated with the GO-Ag nanocomposite. The bacter-
morphology by sonication of the polyvinyl alcohol with ial growth of the cells treated with foils coated with GO
Ag-NP, GO, and GO-Ag NP solutions on the foils. Pure and Ag-NPs was slightly lower than that of cells in the
polyurethane foil was used as a reference foil in relation control group whereas the growth of bacterial cells
to foils coated by the ultrasonic method. The images in treated with foils coated with GO-Ag was greatly inhib-
Fig. 8 are the AFM phase contrast images made in AC; ited, 88.6% of E. coli, 79.6% of S. aureus, 76.5% of S. epi-
additionally, cross sections of the GO flakes are at- dermidis,and77.5%ofC.albicans (Figs.9,10,and11).
tached under the corresponding images. Figure 8a is an
image of pure polyurethane film; Fig. 7b depicts the MembraneIntegrity
Ag-NP-coated film, where characteristic and homoge- In cases where the cell membrane is damaged, intracel-
neous grid structures are observable, being similar to lular LDH molecules could be released into the culture
medium. The LDH level outside the cells demonstrates
the cell membrane integrity. Foils coated with GO, Ag-
Table3Testresultsofzetapotential
NPs, and GO-Ag disrupted cell membrane functionality
Sample ZPbyLDE[mV]±SD
and integrity with significant differences between control
Ag-NPs(100μg/mL,afterUS) −5.9±0,4
groups and the Ag-NPs and GO-Ag groups (Fig. 12).
GO(200μg/mL,afterUS) −41.0±3 The highest disruption of cell membranes was observed
GO(200μg/mL)-Ag(100μg/mL,afterUS) −7.1±0,5 in the GO-Ag groups, 66.3% of E. coli, 59.4% of S. aur-
ZPzetapotential,LDElaserDopplerelectrophoresis,SDstandarddeviation,US eus,54.8%ofS.epidermidis,and48.5% ofC.albicans.
ultrasoundmethod,Ag-NPs(asreceived)HydroSilver1000,Amepox,Poland,material
wasnotprocessedAg-NPs(100μg/mL,afterUS)asabovebutprocessedunderUS
ROSProduction
undersimilarconditionstothefoilsintheprecedingsections,GO(asreceived)
materialnotprocessed,GO(200μg/ml,afterUS)asabovebutprocessedunderUS Low levels (or optimum levels) of ROS play an import-
undersimilarconditionstothefoilsintheprecedingsections,GO(200μg/mL)-Ag
ant role in signaling pathways. However, when ROS pro-
(100μg/mL)materialsmixedtogetherandprocessedunderUSundersimilar
conditionstothefoilsintheprecedingsections duction increases and overwhelms the cellular
Jaworskietal.NanoscaleResearchLetters (2018) 13:116 Page10of17
Fig.7Scanningelectronmicroscopyimagesofa,bnon-coatedpolyurethanefoilwithasmoothsurfacewithsingleimpurities;c,dfoilcoated
withGO,thebroken-downGOflakesdepositedonthepolyurethanefoilsurface;e,ffoilcoatedwithAg-NPsonwhichgridstructurescomposed
ofpolyvinylalcoholandAg-NPsareobserved;andg,hfoilcoatedwithGO-Ag,whichwasmixedundertheinfluenceofultrasoundsanddeposited
onthefoilsurface
antioxidantcapacity,itcaninducemacromoleculardam- an end, and new antibacterial agents are needed. In re-
age (by reactingwith DNA, proteins, and lipids) and dis- cent years, studies have reported nanoparticles as a
rupt thiol redox circuits. Foils coated with Ag-NPs and promising alternative to antibacterial reagents because
GO-Ag (P<0.05) increased the ROS production of all of their antibacterial activity in several biomedical appli-
tested microorganisms compared to the control group. cations [19, 45]. Nanoparticles canbe an effective way to
Foils coated with GO only increased the ROS produc- control many pathogenic and antibiotic-resistant micro-
tion ofC.albicans.ThehighestROSproduction wasob- organisms. Among many metal nanoparticles, Ag-NPs
servedintheGO-Aggroup (Fig. 13). havebeen intensely studied because of the distinct prop-
erties of their antibacterial activity [7]. Ag-NPs have
Discussion been proved effective against over 650 microorganisms
The discovery of antibiotics, natural products produced including bacteria (both gram-positive and negative),
by microorganisms that are able to prevent the growth fungi, and viruses; however, the precise mechanism of
of bacteria and thus cure infectious diseases, revolution- antimicrobial action is not understood completely [46].
ized medical therapy; however, the overuse and misuse Ag-NP exposure to microorganisms could cause adhe-
of antibiotics have been key factors contributing to anti- sion of nanoparticles to the peptidoglycan and the cell
biotic resistance. Now, theeraofantibiotics iscomingto membrane [47], penetration inside the cell [48],