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Graphene oxide–silver nanocomposites modulate
biofilm formation and extracellular polymeric
substance (EPS) production†
Shima Liu,a,b Shuting Cao,b Jingyang Guo,a,b Liqiang Luo,*a Yi Zhou,c Chenglie Lin,c
Jiye Shi,d Chunhai Fan, b Min Lv*b and Lihua Wang *b
Biofilms with positive and negative actions ubiquitously affect medical infections, environmental remediation and industrial processes. However, it remains challenging to control the growth of harmful biofilms
as well as to exploit the use of beneficial biofilms. Here we investigated the effect of an antibacterial graphene oxide–silver nanoparticles (GO–AgNPs) composite on Pseudomonas aeruginosa biofilm formation. We found that GO–AgNPs prevented biofilm formation in a dose-dependent manner, with a
threshold of 15 μg mL−1. Interestingly, the bacterial biomass significantly decreased, but extracellular polymeric substance (EPS) production remarkably increased in mature biofilms treated with GO–AgNPs of an
Received 19th May 2018,
Accepted 17th September 2018
appropriate concentration, suggesting that GO–AgNPs effectively modulate biofilm development and
structure. Moreover, we established that GO–AgNPs caused bacterial death via both physical damage and
DOI: 10.1039/c8nr04064h
oxidative stress, showing the synergic action of GO and AgNPs. These findings facilitate the use of gra-
rsc.li/nanoscale
phene-based nanocomposites for greener antibiotic applications.
Introduction
Biofilms have attracted widespread attention owing to their
significant impacts on medical infections, environmental
remediation and industrial processes.1–3 Bacteria in nature
and artificial systems tend to form surface associating bacterial communities embedded in self-produced three-dimensional extracellular polymeric substances (EPS), called biofilms. The typical biofilm formation starts from planktonic
bacteria adhesion, subsequent cell growth and EPS production. The key components of EPS include polysaccharides,
proteins, lipids and extracellular DNA (eDNA).4 The hydrogellike EPS protects bacteria within a biofilm from ambient
insults and attacks, such as antibiotics, chemicals and heterogeneous bacteria, resulting in strong disinfection-resistance.5
To date, biofilm-associated infections have been a major threat
a
College of Sciences, Shanghai University, Shanghai 200444, China.
E-mail: luck@shu.edu.cn
b
Division of Physical Biology & Bioimaging Center, Shanghai Synchrotron Radiation
Facility, CAS Key Laboratory of Interfacial Physics and Technology, Shanghai
Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China.
E-mail: wanglihua@sinap.ac.cn, lvmin@sinap.ac.cn
c
School of Basic Medicine, Chengdu University of Traditional Chinese Medicine,
Chengdu 611137, China
d
UCB Pharma, Slough, Berkshire SL1 3WE, UK
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c8nr04064h
This journal is © The Royal Society of Chemistry 2018
to public health globally. It has been estimated that 80% of all
infectious diseases are caused by biofilm-associated microorganisms that are the fourth major cause of death, and it
costs more than 1 billion dollars annually to treat these
biofilm-based infections.6 However, these sessile communities
already proved to possess beneficial applications, such as
wastewater treatment, medical application and novel bioengineered materials preparation based on sticky biopolymer-containing EPS.2,3,7 The increase of biofilm biomass, especially
EPS in a sludge blanket system, is a highly effective strategy for
the removal of soluble biodegradable pollutants.2 Moreover,
EPS with important physiochemical and biological properties
has promising applications in medical and biomedical areas,
such as tissue scaffolds, drug delivery, and anti-inflammatory
agents.1,3 Given these positive and negative features of biofilms, it is important to control the harmful biofilms as well as
to exploit the beneficial biofilms. Nanomaterials with excellent
antibacterial activity and specific antimicrobial mechanisms
have been emerging as strong candidates for traditional antimicrobial agents in recent years.8 Many studies reported that
these antibacterial nanomaterials could not only kill planktonic bacteria, but could also inhibit biofilm formation and
even eliminate mature biofilms,9 such as single-wall carbon
nanotubes,10 vanadium pentoxide nanoparticles,11 and zinc
oxide nanoparticles.12 Nevertheless, there was much debate
about their broad applications in clinics and industry due to
their poor stabilities and potential toxicities to humans and
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the environment.13 Graphene oxide (GO) as a novel antibacterial material has attracted great attention in biomedicine over
the past few decades because of its excellent capacities against
bacteria and biocompatibility.14–17 GO is a single-layer sp2
hybridized carbon network with abundant functional groups
including hydroxyl, carboxyl and epoxide groups, possessing a
large surface area, highly specific mechanical properties, and
especially good water dispersibility. These characteristics make
GO thoroughly to come into contact with bacteria and disrupt
their cellular membrane in aqueous systems, presenting
superior antibacterial activity to other graphene derivatives.15
In addition, GO with various defects and functional groups is
an ideal platform to fabricate nanocomposites, which provides
insights into the preparation of novel antimicrobial
agents.18,19 Recent studies focused on the fabrication and
application of graphene oxide–silver nanoparticle composites
(GO–AgNPs).20 AgNPs are known to be antiseptic to a broadspectrum of bacteria, have low drug-resistance and good biocompatibility in nanobiomedicine.21,22 However, the agglomeration of AgNPs decreases the antimicrobial capacity, and
even makes Gram-negative bacteria become resistant to their
toxic effect.23,24 Previous studies found that the GO component
can effectively prevent the aggregation of AgNPs in GO–AgNPs
composites, showing an enhanced inhibition activity compared to pure GO or AgNPs. To date, many studies have mainly
focused on the broad-spectrum inhibition of GO–AgNPs
against planktonic microorganisms. Actually, bacteria inhabit
natural and artificial systems in the form of homogeneous or
heterogeneous biofilms rather than planktonic individuals.
The community has improved tolerance to antimicrobial
Nanoscale
agents compared to individual bacteria. Thus, it is crucial to
study the impact of GO–AgNPs on the development of
biofilms.
In this study, we employed Gram-negative bacteria
Pseudomonas aeruginosa (P. aeruginosa) as a model organism to
investigate the effect of GO–AgNPs on the biofilm formation. A
P. aeruginosa biofilm plays an important role in nosocomial
infections such as burn wounds and cystic fibrosis (CF).25 We
prepared GO–AgNPs composites by in situ reduction of Ag+ to
AgNPs on the GO surface, and systematically examined the
effect of GO–AgNPs treatments with different concentrations
and processing times on the development and structure of biofilms. These findings provided insights into the application of
GO–AgNPs for the modulation of biofilms.
Results and discussion
Synthesis and characterization of GO–AgNPs
To prepare GO–AgNPs, a GO nanosheet was synthesized
according to modified Hummers’ method.14,26 Then, AgNPs
were grown on the GO surface via reducing silver ions.24
Transmission Electron Microscopy (TEM) was used to observe
the morphology of GO–AgNPs. The images showed that
uniform Ag nanoparticles were decorated on single-layer GO
(Fig. 1a & b). The bare GO was about 1 nm thick and has a
lateral size in a range from hundreds of nanometers to two
micrometers (Fig. S1†). As shown in Fig. 1d, the size range of
AgNPs was from 2 to 10 nm by randomly measuring 1504 Ag
nanoparticles of 10 TEM images, with an average diameter of
Fig. 1 Characterization of the GO–AgNPs composite. (a, b) Typical TEM images of GO–AgNPs (a, zoom out, b, zoom in). (c) HRTEM image of a
single Ag nanoparticle and (d) size distribution of AgNPs decorated on GO nanosheets. (e) UV-vis absorption spectra and (f ) the XRD spectrum of
GO and GO–AgNPs.
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7.06 ± 2.54 nm. The high-resolution TEM (HRTEM) image of
the composite showed in the spherical shaped AgNPs with a
polycrystalline structure with an inter-planar spacing (d
spacing) of 0.237 nm, which was in line with the (111) l d
spacing reported in the Joint Committee Powder Diffraction
Standards (JCPDS, no. 04-0783).27 UV-vis spectroscopy was
carried out to analyze the bonds in GO–AgNPs (Fig. 1e). The
absorption spectrum of the GO sheets showed a featured peak
at 220 nm, which was attributed to the π–π* transitions of the
aromatic CvC bonds.28 However, a new peak appeared at
402 nm, which was attributed to the surface plasmon resonance (SPR) of the spherical AgNPs, indicating the presence of
AgNPs on the GO–AgNPs surface.29,30 Inductively coupled
plasma-mass spectrometry (ICP-MS) analysis provided
additional quantitative evidence for the presence of 1.28 μg
mL−1 Ag element in 10 μg mL−1 GO–AgNPs.
X-ray diffraction (XRD) was carried out to analyze the crystal
structure of pristine GO–AgNPs (red line) and GO (black line).
The GO had a sharp peak at 10.8°,31 which was assigned to the
(002) inter-planar spacing of 0.82 nm. The XRD spectrum of
the GO–AgNPs composite showed typical peaks at 2θ = 38.3°,
44.4°, 64.6°, and 77.6°. These peaks were related to the (111),
(200), (220), and (311) crystalline planes of the face-centered
cubic Ag crystals, respectively, revealing that GO–AgNPs had
been obtained.24,29 Moreover, the d spacing of the (111) plane
for Ag was 0.235 nm, the same as the HRTEM result. However,
the typical diffraction peak at 2θ = 10.8° decreased remarkably
in the GO–AgNPs group, which indicated that AgNPs protected
GO layers from self-stacking. The positively charged Ag ions
can bind with the negatively charged oxygen-containing functional group of GO via an electrostatic force and be reduced
into Ag nanoparticles, which may disrupt the regular layer
structures of GO and expand the restacked GO.32,33 These results
suggested the successful synthesis of GO–AgNPs composites.
Inhibition of the biofilm formation by GO–AgNPs
To investigate the effect of GO–AgNPs on P. aeruginosa cell
growth and biofilm formation, the optical density method
(OD600) based on the turbidity of the bacterial suspension,
and crystal violet staining were used, respectively. As shown in
Fig. 2, P. aeruginosa growth and biofilm formation decreased
dose-dependently after 12 h exposure to GO–AgNPs at concentrations varying from 10 to 25 μg mL−1 under static conditions,
suggesting the inhibition of proliferation of P. aeruginosa. The
number of bacteria and the biomass of the biofilm both
decreased more than 50% in the presence of 10 μg mL−1 GO–
AgNPs. At a concentration above 15 μg mL−1, GO–AgNPs completely inhibited biofilm formation. We further confirmed this
effect by a combination of fluorescence-based confocal images
and computational analysis.34–36 Bacterial cells and EPS of biofilms were labelled with SYTO9 (in green) and concanavalin
A-Alexa Fluor 647 conjugate (in red), respectively. The results
showed that the biomass of the control biofilm containing bacteria and EPS was about 3 and 50 times higher than the 15
and 25 μg mL−1 GO–AgNPs group, respectively (Fig. 2d–g &
Table 1). The coverage of the biofilm without and with 15 and
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25 μg mL−1 GO–AgNPs was 99.20 ± 1.60%, 40.80 ± 3.78% and
5.92 ± 1.76% (Table 1). The substratum coverage was defined
as the area coverage in the first layer of cell adhesion and proliferation on the substratum.10 Therefore, the reduction of the
biofilm could be explained by the fact that GO–AgNPs
decreased the adhesion number of the planktonic cell at the
initial stage by preventing the growth and proliferation of
planktonic P. aeruginosa, and even by inactivating all cells at
high concentrations (Fig. 2a).
Importantly, GO–AgNPs exhibited remarkably enhanced
antibacterial properties compared to bare GO or AgNPs when
the amount of GO (21.8 μg mL−1) and AgNPs (3.2 μg mL−1) was
approximately equal to that in 25 μg mL−1 GO–AgNPs. As
shown in Fig. 2c, both GO and AgNPs had a little influence on
the biofilm formation, but GO–AgNPs significantly inhibited
biofilm formation. The results showed that P. aeruginosa
biofilm formation was fully inhibited by pure GO and AgNPs
at the concentrations of 200 μg mL−1 and 8 μg mL−1, respectively (Fig. S3†). The excellent inhibition effect of the GO–
AgNPs against bacterial growth and biofilm formation could
be ascribed to the synergistic effect of the AgNPs and GO,
named the “capturing–killing process”.29,37 Single-layer GO
sheets have stronger adsorption properties owing to their large
specific surface area and negatively charged oxygen-containing
groups, therefore the bacterial cells may be adsorbed onto
their surfaces, which promotes the interaction between the
bacteria and nanocomposites. In addition, AgNPs are
anchored on the surface of GO, effectively preventing their
aggregation to maintain high surface reactivity and outstanding antibacterial properties.29
Dynamics of cell growth and biofilm formation
To further explore the anti-biofilm formation of GO–AgNPs, we
observed the dynamics of biofilm development up to 36 h.
Biofilms develop through a series of stages. First, planktonic
cells attach to the surface. Second, bacteria grow and secrete
EPS to form mature three-dimensional communities. Finally,
the single cells from the mature biofilm migrate to new surfaces, which was also considered as the beginning of a new life
cycle (Fig. 3a). Fig. 3b shows the control floating cells attached
to the surface and the mature 3D structure after 2 and
12 hours, respectively. However, the adhesion of planktonic
bacteria treated with 15 μg mL−1 GO–AgNPs delayed to 6 h,
and cell proliferation and biofilm maturation extended to
24 h. Interestingly, the maximum biomass of the biofilm
exposed to 15 μg mL−1 GO-AgNPs at 24 h is similar to that of
the control sample at 10 h, which indicated the recovery of bacterial growth.10,38 Bacteria can survive in the presence of bactericidal antibiotics as non-growing or slow-growing, which is
defined as “tolerance”.39 When the antibacterial concentration
is lower than a threshold tolerated by microorganisms, slowgrowing bacteria occur in the part of a clonal population,
resulting in an extended lag time or cell recovery.40 After 24 h,
the biofilm significantly decreased due to the mature biofilm
dispersal and reached the saturation mode at ∼36 h. In contrast, P. aeruginosa biofilm exposure to 25 μg mL−1 GO–AgNPs
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Fig. 2 Effects of GO–AgNPs on the biofilm formation. (a) The bacterial growth and (b) biofilm formation in the presence of different concentrations
of GO–AgNPs. (c) The comparison of the inhibition efficacy of GO–AgNPs to GO and AgNPs. The concentration of AgNPs, GO, and GO–AgNPs was
3.2 μg mL−1, 21.8 μg mL−1 and 25 μg mL−1, respectively. *** indicate significant differences at p < 0.001. (d) The confocal images of the biofilm structure. Live cells stained with SYTO9 dye (green), and EPS stained with Con-A Alexa Fluor (red). Scale bar, 100 μm. Bacteria and EPS across the biofilm
thickness under the different culture conditions. (e) Control, (f ) 15 μg mL−1 and (g) 25 μg mL−1 GO–AgNPs. The insets of (b) and (c) are the digital
images of the biofilm stained CV.
Table 1 Confocal analysis of biofilm growth for 12 h in media with
GO–AgNPs
Analyses
Control
15 μg mL−1
25 μg mL−1
Total biomass (μm3 μm−2)
Cell biomass (μm3 μm−2)
EPS biomass (μm3 μm−2)
Substratum coverage (%)
Average thickness (μm)
Roughness coefficient
27.88 ± 4.68
26.28 ± 4.13
1.46 ± 0.45
99.20 ± 1.60
35.42 ± 1.53
0.12 ± 0.04
9.13 ± 2.10
8.60 ± 1.51
0.53 ± 0.13
40.80 ± 3.78
14.13 ± 4.23
0.88 ± 0.19
0.58 ± 0.07
0.46 ± 0.05
0.12 ± 0.01
5.92 ± 1.76
0.63 ± 0.08
0.78 ± 0.22
Data are shown as mean ± standard deviation.
was totally inhibited. The growth of total and suspended bacterial cells showed a similar tendency to biofilm formation.
The planktonic bacterial growth curve includes the lag, log
and stationary phase. As shown in Fig. 3d, control bacteria
grew and reached the stationary phase after 14 hours OD600
(1.35) compared to after 24 hours OD600 (1.16) in the case of
15 μg mL−1 GO–AgNPs. Remarkably, the growth of bacteria
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was entirely inhibited during the whole 36 h culture period
when the concentration of the GO–AgNPs was increased to
25 μg mL−1. These results suggested that GO–AgNPs have a
threshold tolerated by P. aeruginosa like other antibacterial
agents.10 At concentrations lower than threshold, the toxicity
of the GO–AgNPs to bacteria may occur only temporarily, and
then the cells restore normal growth and biofilm formation.
The GO–AgNPs concentration over 15 μg mL−1 caused complete inhibition of bacterial growth and biofilm formation.
Thus, the biofilm formation could be regulated by selecting
the optimal concentration of GO–AgNPs.
GO–AgNPs exposure affects the mature biofilm structure
CV staining demonstrated that the total biomass including
bacterial cells and EPS of the mature biofilm treated with
15 μg mL−1 GO–AgNPs was close to that of the untreated
group.41 To characterize the detailed structure and component
of biofilms, we used confocal imaging to evaluate the biomass
of bacterial cells and EPS respectively. As shown in Fig. 4a & b,
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Fig. 3 Dynamics of cell growth and biofilm formation. (a) Schematic illustration of the development of biofilms untreated and treated with GO–
AgNPs. (b) CV staining images of biofilms. (c) Dynamics of biofilm formation. (d) The growth curves of the total and suspended bacteria. Bacteria
were exposed to GO–AgNPs to form biofilms for 36 h at a concentration of 0, 15 and 25 μg mL−1, respectively.
GO–AgNPs addition significantly decreased bacterial cells ( p <
0.05) but enhanced EPS production of the biofilm ( p < 0.001)
compared to that of the control sample, which revealed that
GO–AgNPs with antibacterial properties stimulated bacteria’s
resistance to harsh environments through EPS overproduction.
We carried out SEM to observe the bacterial morphology and
biofilm structure. The control and the P. aeruginosa biofilm
exposed to 15 μg mL−1 GO–AgNPs were similarly fully covered
with multilayer P. aeruginosa biofilms, with a thickness of
∼6.14 μm and ∼6.80 μm, respectively (Fig. 4c), which is consistent with the quantitative analysis of confocal images
shown in Table S1.† However, we found a remarkable increase
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of EPS secreted by the bacteria exposed to GO–AgNPs, providing a more compact structure than the control as shown in
Fig. 4c (top-view). These findings indicated that the optimal
concentration of GO–AgNPs could effectively prevent the proliferation of bacteria but improve EPS production, which
benefits wastewater treatment in industry.
The mechanism of GO–AgNPs action
Graphene-based nanomaterials have been proved to cause
membrane disruption due to their strong interaction with bacteria.14,42 As shown in Fig. 5, the change of the bacterial morphology when treated with GO–AgNPs was dose-dependent.
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Fig. 4 Biofilm structure in the mature stage. (a) The confocal images and analysis of the biofilm. Live cells were stained with SYTO9 dye (green),
and EPS was stained with Con-A Alexa Fluor (red). Scale bar, 100 μm. Bacteria and EPS across the biofilm thickness under the different culture conditions. (i) Control, (ii) 15 μg mL−1 GO–AgNPs. (b) The biomass of the biofilm components containing bacteria and EPS. (c) SEM images of the biofilm
in the top and side view respectively. Scale bar, 1 μm.
Few cells were damaged in the biofilm in the presence of
15 μg mL−1 GO–AgNPs (Fig. 5b), explaining the recovery of bacterial growth and biofilm formation. In contrast, most of the
bacteria lost the integrity of the cellular membrane (Fig. 5c &
d, white arrow). Thus, physical damage induced by the interaction between the blade-like graphene composites and bacteria is responsible for the decrease of adhesion bacteria in
the development of biofilms. It is difficult to measure the
thickness of a biofilm by SEM (Fig. S4†). Besides, ROS is considered as another universal mechanism in nanomaterial toxicity.15,43 We carried out an ESR technique with DMPO to
ascertain the generation and species of ROS.44,45 A hydroxyl
radical (•OH) reacts stably with DMPO to form DMPO-•OH
adducts in water. Nevertheless, a superoxide radical (•O2−) is
very unstable in water and undergoes facile disproportionation, which reacts with DMPO to generate DMPO-•O2− adducts
in ethanol. It has been reported that DMPO-•OH possesses
four characteristic peaks with an intensity of 1 : 2 : 2 : 1,44 but
they were not observed in our experiment (Fig. 6a). GO–AgNPs
and GO gave six characteristic peaks with intensities similar to
the spectra reported in the previous studies of DMPO-•O2−
adducts. However, no typical signal was found in AgNPs.
These ESR results confirmed that •O2− radicals existed in GO–
based nanomaterials. Previous studies reported that the superoxide radical attacks the lipids and proteins of the cellular
membrane, which results in the loss of cellular integrity and
Fig. 5 SEM images of bacterial morphologies in biofilms with and without GO–AgNPs. (a) Control (b) 15 μg mL−1 and (c) 25 μg mL−1. (d) High-magnification images of (c) (the indicated portion).
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Fig. 6 ROS generation and the effect of nanomaterials. (a) DMPO spin-trapping EPR spectra detected ROS production. (b) Oxidation of GSH by antibacterial nanomaterials, 3.2 μg mL−1 AgNPs, 21.8 μg mL−1 GO and 25 μg mL−1 GO–AgNPs. The positive control is 1 mM H2O2. Data are represented
as mean ± s.d. * indicates the significant difference at p < 0.05 compared with the control. **p < 0.01, ***p < 0.001.
the death of cells. Thiol groups of these biological molecules
are very susceptible sites for attack by ROS.46 Thus, we employed
Ellman’s assay to confirm the oxidation capacities of these
nanomaterials.15 Fig. 6 shows that 28.57 ± 0.37%, 22.20 ± 0.23%
and 4.47 ± 2.21% of GSH was oxidized by GO–AgNPs, GO and
AgNPs, respectively. GO–AgNPs showed superior oxidation
activity to AgNPs, but similar to GO. Thus, the generation of
ROS by GO–AgNPs here possibly resulted from GO and finally
caused bacterial membrane disruption and activity loss.
Given the harmful and useful actions of bacterial biofilms,
it is important to regulate their development. On the one
hand, various antibacterial agents are widely used to prevent
biofilm growth, such as antibiotics, metal ions and nanomaterials. On the other hand, researchers have devoted their
efforts to improving the production of EPS for wastewater treatment and new bioengineered material fabrication based on a
biopolymer-based sticky structure.1,2 The typical biofilm develops through several stages, including planktonic bacteria
adhesion, aggregation and growth, biofilm formation, maturation and dispersal (Fig. 3a). In our study, the addition of
GO–AgNPs would first encounter planktonic cells and subsequently participate in the formation of a biofilm. The floating bacteria in the medium significantly reduced in a dosedependent manner after GO–AgNPs addition, resulting in the
biofilm formation inhibition. The exponential and stationary
stages of the planktonic cells were delayed in the presence of
GO–AgNPs at a concentration of 15 μg mL−1. The development
of treated biofilms also lagged behind the untreated sample.
Above the concentration of 15 μg mL−1, GO–AgNPs killed
almost all planktonic bacteria at the initial stage, leading to no
biofilm formation. Moreover, the combination of Ag nanoparticles and GO remarkably improved the antimicrobial and
anti-biofilm activities compared to pure GO or AgNPs. Physical
damage and oxidative stress are the two most common mechanisms of nanomaterial toxicity toward microorganisms. The
generation of ROS triggered by both GO and AgNPs has been
considered as a primary mode of toxic action.47,48 A very high
oxidative stress often causes bacterial metabolic disorder and
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cellular membrane disruption, leading to inactivation or even
death of bacterial cells.22,43 On the other hand, both GO and
AgNPs can destroy the cellular membrane by physical interactions, but different mechanisms of action were illustrated.
AgNPs with a large specific surface area tend to adsorb and
accumulate on the bacterial surface when brought into contact
with bacteria, causing the loss of bacterial membrane integrity
and cellular death.48 However, single-layer GO with sharp
edges can penetrate the microbial cellular membrane, and
then seriously damage the cellular membrane and result in
the leakage of the cell content and the final death of bacteria.14,16,49 Besides, each of them have their own specific antibacterial mechanism. For example, Ag ions (Ag+) released from
AgNPs play an important role in bacterial inactivation.28,31 GO
nanosheets with flexible properties and strong absorption
enable them to wrap the bacterial surface and block the
exchange of substances inside and outside of cells, inhibiting
bacterial growth and proliferation.50 The enhanced antibacterial properties of GO–AgNPs may result from the synergistic
effect of GO and AgNPs described as the “capturing–killing
process”.29,51 The GO–AgNPs composite like the other two
nanomaterials has strong absorption capacity for the bacterial
cells owing to its high surface area, which improves the
contact between the bacteria and Ag nanoparticles.19,49,52
AgNPs anchored on the surface of GO effectively overcame the
drawback of agglomeration, improving their antibacterial
activity. AgNPs could trigger the death of bacteria through
destroying the cellular integrity, division and function of respiration, because of their interaction with sulfur-containing
proteins and DNA.21 Additionally, GO–AgNPs generated •O2−
radicals and oxidized GSH, suggesting potential oxidative
stress to cells. Although previous studies reported that AgNPs
can inactivate bacteria through oxidative stress, we did not
detect ROS production by itself. The oxidative capability of
GO–AgNPs should be mainly from GO that can mediate the
production of ROS by the adsorption of O2 on its defect sites
and edges. Taken together, direct killing of bacteria was the
main reason for the inhibition of biofilm formation in the
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presence of GO–AgNPs. Kulshrestha et al. reported that GO–
AgNPs led to the down-regulation in the gene expression of
Streptococcus mutans (S. mutans) including comDE, spaP and
virR. These genes played crucial roles in adherence and the
quorum sensing cascade of S. mutans.53 We noted that GO–
AgNPs inhibited the adhesion of bacteria and proliferation,
but also improved EPS secretion. Our findings suggested that
GO–AgNPs could be potentially applied as a regulator for
biofilm development as well as EPS production.
Conclusions
In this study, we provided a new strategy for regulating bacterial growth and biofilm formation. At the set time (12 h), the
inhibition of GO–AgNPs showed characteristic dose-dependent
behavior. GO–AgNPs exhibited a stronger inhibition activity
than pure GO and AgNPs, which was attributed to the synergic
action between GO and AgNPs. Interestingly, the dynamics of
bacterial growth and biofilm formation found that GO–AgNPs
had a threshold of 15 μg mL−1 tolerated by P. aeruginosa. GO–
AgNPs at this concentration made the bacterial growth and
biofilm development slower, but both of them could recover
the normal total biomass at the mature stage. Surprisingly, the
bacterial biomass significantly decreased, but EPS biomass
notably increased. Above this concentration, biofilm formation
was completely prevented, which could be attributed to physical damage and oxidative stress by GO–AgNPs. The effects of
GO–AgNPs on the biofilm not only provided insights into the
application of GO-based nanomaterials, but also into biofilm
modulation in industrial and clinical treatments.
Experimental section
Sample preparation, characterization and analysis can be
found in the ESI.†
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This work was supported by the National Key R&D Program of
China (2016YFA0201200, 2016YFA0400900) and the National
Natural Science Foundation of China (U1432116, 11675251,
61571278).
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