Carbon 105 (2016) 362e376

Contents lists available at ScienceDirect

Carbon
journal homepage: www.elsevier.com/locate/carbon

Review article

The controversial antibacterial activity of graphene-based materials

Hanaa M. Hegab a, b, Ahmed ElMekawy c, d, Linda Zou a, e, Dennis Mulcahy a,
Christopher P. Saint a, Milena Ginic-Markovic f, *
a
Centre for Water Management and Reuse, University of South Australia, Adelaide, SA 5095, Australia
b
Institute of Advanced Technology and New Materials, City of Scientific Research and Technological Applications, Borg Elarab, Alexandria, Egypt
c
Genetic Engineering and Biotechnology Research Institute, University of Sadat City (USC), Sadat City, Egypt
d
School of Chemical Engineering, University of Adelaide, Adelaide, SA 5005, Australia
e
Department of Chemical and Environmental Engineering, Masdar Institute of Science and Technology, Abu Dhabi, United Arab Emirates
f
Future Industries Institute, University of South Australia, Adelaide, SA 5095, Australia

a r t i c l e i n f o a b s t r a c t

Article history: Graphene (Gr)-based materials are a promising nanomaterial for the development of antibacterial sur-
Received 11 January 2016 faces owing to their biocidal activity. However, the effect of the physicochemical features of these ma-
Received in revised form terials on their antibacterial activity has yet to be clarified. Gr-based nanomaterials can interact with
18 April 2016
cellular components, e.g. membranes, proteins and DNA, and initiate a sequence of nanomaterials/bac-
Accepted 19 April 2016
Available online 23 April 2016
terial interactions that rely on colloidal energies and active bio-physicochemical interfaces. Analyzing
these different interfaces permits the development of anticipated relations between physical/chemical
structure and bactericidal activity depending on Gr-based nanomaterial features such as shape, size,
hydrophilicity, roughness and functionality. Realizing how nanomaterials are interacting with bacterial
cell membranes is correlated to how they affect bactericidal activity and is thus critical for obtaining
benign applications. This review analytically discusses specific Gr-based material features related to
bacterial interactions, with special focus on the different modes of interaction between Gr-based ma-
terials and cell membranes, nucleic acids and lipid bilayers.
© 2016 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363
2. Antibacterial activities of Gr-based materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365
2.1. Gr-based nanocomposite dispersions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365
2.2. Gr-based nanocomposite surfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366
2.3. Gr-based hydrogels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366
2.4. Bacterial reduction of GO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367
3. Gr-based materials features related to biocidal activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367
3.1. Sheet size and concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367
3.2. Surface area . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367
3.3. Surface roughness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 368
3.4. Hydrophilicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 368
3.5. Dispersibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 368
3.6. Functionalization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369
4. Antibacterial mechanisms of Gr-based materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369
4.1. General mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369
4.1.1. Bacterial membrane stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369
4.1.2. Oxidative stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369
4.1.3. Wrapping isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371

* Corresponding author.

http://dx.doi.org/10.1016/j.carbon.2016.04.046
0008-6223/© 2016 Elsevier Ltd. All rights reserved.
 H.M. Hegab et al. / Carbon 105 (2016) 362e376 363

4.2. Gr-based suspension/membrane antibacterial capabilities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371

5. Potential of Gr antiviral ability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373
6. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373

1. Introduction adherence of Gr to biological cells [10].

When the toxicity of Gr nanosheets against bacteria and fungi
Biocidal nanomaterials is an attractive field of science that has was investigated, it was observed that these nanosheets can
the capability to, amongst other things, enhance medical devices possibly be applied as antimicrobial products, in a way similar to
[1], wastewater treatment [2], food packaging [3], synthetic textiles the most commonly used antimicrobial nanoscale materials like
[4] and dentistry [5] (Fig. 1). Development of novel and efficient silver nanoparticles (Ag NPs) [11]. The widespread use of Gr
antibacterial agents is urgently required due to the growing anti- nanosheets in biological applications is a result of their outstanding
bacterial resistance spectrum of bacterial infections. The antibac- bactericidal activity on a number of Gram negative and positive
terial action of nanoparticles (NPs) is generally assessed against the bacteria. This has increased the attraction towards the synthesis of
typical group of pathogenic bacteria, e.g. Staphylococcus aureus, GO along with its derivatives and rGO [12]. It has been observed
Escherichia coli, Listeria monocytogenes, Streptococcus mutans and that the degree of antibacterial effect of nanomaterials is deter-
Pseudomonas aeruginosa, which are responsible for different hu- mined by their shape, surface functionalization, size, stability and
man infections related to weakened immunity and are a significant size distribution [6]. Consequently, several GO nanosheets cova-
threat to public health [6]. Accordingly, developing nanomaterial lently and non-covalently functionalised were developed in order
surface coatings with effective antibacterial abilities is vital for to improve their antibacterial effect, stability and decrease their
human health and wellbeing. One of the most recently developed negative ecophysiological toxic side effects [13e15].
biocidal nanomaterials is graphene (Gr), which has been inten- The bactericidal effect of Gr-based materials is controversial and
sively studied as a carbon based nanomaterial. It can be considered requires further analysis. For example, in one study the antibacte-
as a single atom sheet of graphite (Gt). Gr oxide GO nanosheet is the rial property of four kinds of carbon-based materials against E. coli
oxidized form of Gr. It is a two-dimensional, one atom-thick sheet was compared [16]. GO was observed to display the maximum
with sp2-carbon atoms organized hexagonally and ornamented antibacterial activity towards E. coli as compared to Gt, Gr and rGO.
with several oxygen functional groups. Likely, it is the slimmest Another study was conducted to investigate the antimicrobial ac-
alignment of carbon molecules and is a key element for other tivity of GO [17], and results revealed faster bacterial growth, when
graphitic derivatives, Fullerenes and carbon nanotubes (CNTs) [7]. the culture medium was supplemented with GO. This was
Gr has exceptional physicochemical features, which include robust explained by the stimulation of bacterial proliferation via GO,
mechanical strength, great surface area and resistance to degra- which acts as a surface for cellular attachment and growth. Such
dation [7,8], due to its superior surface-to-volume ratio [9]. The significant outcomes for the application of Gr-based materials as
active segments, edges and surfaces, enable the attachment and antibacterial agents are a direct consequence of the intensive
attention these nanomaterials have attracted over the last six years,
as apparent from the number of publications on this topic. Fig. 2
demonstrates that searching ‘ScienceDirect’, using the keywords
‘‘graphene and antibacterial”, showed 96% growth in the total sci-
entific publications over the previous six years (2009e2015). More
than half of this research was conducted in the last two years.
Accordingly, in the present review the most contemporary

Fig. 1. Potential applications of antibacterial graphene in different fields including Fig. 2. The scientific research progress in biocidal activity of graphene based on the
water purification, energy and medicine. (A colour version of this figure can be viewed number of articles in the “ScienceDirect” database till November 2015. (A colour
online.) version of this figure can be viewed online.)
364 H.M. Hegab et al. / Carbon 105 (2016) 362e376

Table 1
The features and antibacterial efficiency of different freestanding and nanocomposite Gr-based materials. (A colour version of this figure can be viewed online.)

Gr-based materials features Antibacterial evaluation

Type Hybrid material Fabrication method Concentration Bacterial strains Incubation Method Inhibition Reference
(mg/ml) time (h) (%)

Gra-based Nanocomposite GOb Hummers and Offeman 175 P.hh aeruginosa 2 PCnn 100 [22]
Dispersions rGOc Hummers and Offeman 102 E.ii coli 4 PC 88 [138]
GO Hummers and Offeman 85 E. coli 2 PC 98.5 [20]
GO Hummers and Offeman 40 E. coli 2 PC 69.3 [16]
GO Hummers and Offeman 40 E. coli 2 PC 97.7 [21]
Gr QDsd Electrochemical 200 E. coli/S.jj aureus 0.25 PC 80/92 [23]
Gelatine/Age Green-Synthesis 10 E. coli 24 PC 99.9 [139]
nanoprisms
Ag NPsf Turkevich method with GO 5 P. aeruginosa 0.5 PC 100 [24]
dispersion
2
Ag NPs Mixing 10 E. coli 4 PC 100 [25]
Ag NPs Electrostatic interactions 3.2/6.4 E. coli 24 PC 99.9 [26]
Ag NPs Solution-based N/Agg E. coli/P. aeruginosa 24 ADoo 18/26 mm [27]
Ag NPs Chemical deposition N/A S. aureus/B.kk subtilis 24 AD 100 [28]
Ag NPs Sequential repetitive chemical N/A E. coli 0.3 AD 20 mm [29]
reduction
Ag NPs Supercritical CO2 10 E. coli/S. aureus 24 PC 100 [30]
Ag NPs Mixing 45 E. coli/S. aureus 4 PC 100 [31]
Ag NPs/PDDAg One-pot hydrothermal 50 E. coli 24 PC 100 [32]
Ag NPs/PEIh Mixing 958
103 E. coli/S. aureus 6 PC 14.8/20.5 [33]
Ag NPs/PDAi In situ reduction 25 E. coli 24 AD 23.7 mm [34]
Ag NPs/PAAj In situ grafting N/A E. coli/S. aureus 24 AD 9.9/ [35]
11.4 mm
Ag NPs/ Nucleophilic substitution 500 E. coli/S. aureus 8 PC 100 [140]
aminophenol
Ag/Fe3O4k Hydrothermal N/A E. coli 24 AD N/A [37]
Ag/TiO2l Two-phase 102 E. coli 2 PC 67 [36]
Fe3O4 NPs Mixing 3
102 E. coli 2 PC 91.5 [40]
ZnOm NPs Hydrothermal 6.6
105 Sal.ll typhi/E. coli 24 AD 13/11 mm [41]
ZnO NPs Mixing 3
103 E. coli 12 PC 100 [38]
MnOxn QDs/TiO2 UV excitation/microwave 31.25/15.62 E. coli/S. aureus 18 AD 10.9/ [43]
10.5 mm
MnFe2O4 Solvo-thermal 102 E. coli N/A PC 82 [44]
TiO2 Mixing N/A E. coli 0.5 PC 99.6 [39]
CuNPso/PLLp Mixing 50 E. coli 24 Tpp 99 [45]
Bi2WO6 Hydrothermal 250 Mixed culture 18 PC 100 [141]
Ag/CDq Mixing 0.05 B. subtilis 24 AD N/A [47]
CdSr Solvo-thermal 200 E. coli 1 PC 99.9 [48]
PEGs/PHGCt Mixing N/A E. coli/S. aureus 1 PC 100 [142]
Csu Drop-casting 3
103 P. aeruginosa 24 PC 100 [115]
Dithiothreitol Solvo-thermal 102 E. coli 4 PC 86 [138]
Sand Mixing N/A E. coli 24 AD 20 mm [143]
Ramizol Mixing N/A S. aureus 20 T 100 [144]
Gr-based Nanocomposite Ag NPs/PA Wet chemical reduction 103 E. coli 1 PC 98 [145]
Surfaces Ag/PESv Immersion phase inversion N/A E. coli/S. aureus/P. 24 AD N/A [59]
aeruginosa
Ag NPs/CAw Flow-directed assembly N/A E. coli 2 PC 86 [60]
Ag NPs/PSx Wet-phase inversion N/A E. coli 18 FESEMqq 100 [61]
Cs Solution-casting 6
104 S. aureus 3 PC 77 [50]
PVAy/Cs Electrospinning 4
103 E. coli 24 AD 8.6 mm [51]
PES Phase inversion N/A E. coli 4 PC 71 [55]
PA Surface functionalization N/A E. coli 1 PC 65 [146]
PAz Interfacial polymerization N/A E. coli 24 PC 65 [56]
PPaa LbLff- deposition N/A E. coli 12 PC 64 [57]
PLL/HAbb LbL- deposition 105 E. coli 4 PC 66 [52]
PEcc resin Casting N/A P. aeruginosa 24 AD 15 mm [54]
PA/PLL Hybrid grafting 80 Mixed culture 24 ATPrr 99 [53]
PES Phase inversion N/A E. coli 24 PC 74.88 [58]
ZnO NWs Drop-casting 103 E. coli 1 PC 95 [147]
ZnO NWs Electrophoretic deposition 103 E. coli 1 PC 99.5 [147]
SSdd/rGO Electrophoretic deposition N/A E. coli/S. aureus 1 PC 84/95 [63]
Ti Electrophoretic deposition N/A E. coli/S. aureus 24 PC 68.4/72.9 [62]
TiO2 Deposition N/A E. coli 0.5 PC 60 [49]
Cu Chemical vapor deposition N/A E. coli/S. aureus 24 PC 56/34 [64]
Gr-based Hydrogels BKBee One-pot tube inversion 4
103 E. coli/List.mm 48 T 99.3/91 [66]
hydrogel
BKB/PDA 4
103 E. coli/List. 48 T 99.3/91 [66]
Agarose Mixing N/A E. coli/S. aureus N/A PC 100 [65]
Ag Crosslinking 2.5
103 E. coli/S. aureus 0.5 PC 100 [68]
Ag/PVA Electrochemical reduction N/A E. coli/S. aureus 24 AD 100 [67]
a
Gr: Graphene.
b
GO: Graphene oxide.
 H.M. Hegab et al. / Carbon 105 (2016) 362e376 365

c
rGO: Reduced graphene oxide.
d
QDs; Quantum dots.
e
Ag: Silver.
f
NPs: Nanoparticles.
g
PDDA: Poly (diallyl dimethyl ammonium chloride).
h
PEI: Polyethyleneimine.
i
PDA: Polydopamine.
j
PAA: Poly(acrylic acid).
k
Fe3O4: Iron oxide.
l
TiO2: Titanium dioxide.
m
ZnO: Zinc oxide.
n
MnO: Manganese oxide.
o
CuNPs: Copper nanoparticles.
p
PLL: Poly-L-lysine.
q
CD: Cyclodextrin.
r
CdS: Cadmium sulphide.
s
PEG: Polyethylene glycol.
t
PHGC: Polyhexamethylene guanidine hydrochloride.
u
Cs: Chitosan.
v
PES: Polyethersulfone.
w
CA: Cellulose acetate.
x
PS: Polysulfone.
y
PVA: Polyvinyl alcohol.
z
PA: Polyamide.
aa
PP: Polypropylene.
bb
HA: Hyaluronic acid.
cc
PE: Polyester.
dd
SS: Stainless steel.
ee
BKB: Benzalkonium bromide.
ff
LbL: Layer by layer.
gg
N/A: Not applicable.
hh
P.: Pseudomonas.
ii
E.: Escherichia.
jj
S.: Staphylococcus.
kk
B.: Bacillus.
ll
Sal.: Salmonella.
mm
List.: Listeria.
nn
PC: Plate count.
oo
AD: Agar diffusion.
pp
T: Turbidity.
qq
FESEM: Field-emission scanning electron microscopy.
rr
ATP: Adenosine triphosphate.

reports on the antibacterial activities of Gr-based materials are
addressed, in order to resolve the argument about their antibac-
terial activity. Moreover, their mechanisms of interaction with
bacterial cells are deliberated based on their forms and various
influential factors, e.g. sheet size, surface area and functionality, so
that the contradictory subject of the antibacterial effect of Gr-based
materials is revealed.
2. Antibacterial activities of Gr-based materials
In its comprehensive definition, an antibacterial agent is any
mediator that impedes the proliferation and growth of bacterial
cells. The Gr-based materials could follow the same concept.
Generally, Gr-based materials are chemically (i.e. chemical depo-
sition), electrochemically (i.e. electrophoretic deposition) or hy-
drothermally (i.e. solvo-thermal) hybridized with different
functional materials (i.e. silver nanoparticles (Ag NPs), metal oxides
and polymers) before being applied as antibacterial agents. The
features of most recent antibacterial Gr-based dispersions, mem-
branes and hydrogels are listed in Table 1 and summarized in the
following sections.
2.1. Gr-based nanocomposite dispersions
Exfoliated GO is the ultimate option for the synthesis of Gr
applicable dispersion, as it can be produced in bulk from cheap Gt
powder and can easily generate stable dispersions in different
solvents [18]. GO Solutions in ethylene glycol (EG), water and N-
methyl-2-pyrrolidone (NMP) offer a long lasting stability with high
solubility values, e.g. 8.7 mg/mL for NMP [19]. Accordingly, several
workers investigated the antibacterial effects of Gr-based disper-
sions against different Gram positive and negative bacterial cells.
The first trial was conducted in 2010 when the bactericidal ac-
tivities of two Gr-based derivatives in water dispersions; GO and
rGO nanosheets, were reported. It was shown that these Gr-based
nanomaterials can effectively inhibit the growth of E. coli bacte-
ria, with even complete viability loss (98.5%) when GO was applied
[20]. These positive results opened the door for further studies to
evaluate the Gr-based materials’ toxicity against bacterial cells. The
bactericidal activity of four derivatives of carbon-based materials
(Gt, GtO, GO and rGO) against E. coli were evaluated [16]. A GO
dispersion showed the uppermost activity (69.3%), consecutively
followed by rGO, Gt, and GtO with the same concentration and
under the same incubation conditions. Alternatively, the superior
bactericidal activity of GO (25 mg/mL) was disproved when a bac-
terial culture was supplemented with it [17]. Faster bacterial
growth, through higher optical density, was observed compared to
cultures without GO. According to this study, it was obvious that GO
lacks antibacterial capabilities. The contradictory findings extended
to the effect of the GO sheet size on its antibacterial property, in
which E. coli was used for the evaluation of the bactericidal activity
of homogenous GO sheets dispersion, in which their lateral size
diverges by more than 100 times [21]. The bactericidal activity of
GO sheets against E. coli cells depended on lateral size, where larger
366 H.M. Hegab et al. / Carbon 105 (2016) 362e376
GO sheets showed stronger bactericidal activity compared to
smaller ones, with exposure time and concentration considered as
most effective aspects on bactericidal activities. The larger GO
sheets bring about the majority of cell viability loss after 1 h of
exposure time, and their concentration effectively impacts bacte-
ricidal activity at relatively small concentration (<10 mg/mL).
Otherwise, when bacterial cells were incubated with smaller GO
sheets up to 4 h, the inhibition rate of bacterial cells increased
continuously. The threshold antibacterial concentration of GO was
suggested to be 150e175 mg/mL in which no P. aeruginosa cells were
observed after 4 h incubation with these concentrations [22].
Moreover, the antibacterial effect of electrochemically produced Gr
quantum dots were investigated on S. aureus and E. coli. Bacterial
cells lost their viability (80e92%) by the effect of generated reactive
oxygen species (ROS) upon the photoexcitation of Gr quantum dots
[23].
In order to obtain the synergistic antibacterial effect of some
nanomaterials along with Gr-based materials, an alternative strat-
egy was followed by hybridizing Ag NPs with Gr-based materials.
Several approaches were applied to assemble Ag NPs on GO sheets,
i.e. one-pot hydrothermal, electrostatic interactions, simple mixing,
chemical deposition, sequential repetitive chemical reduction and
supercritical CO2. The resultant nanocomposites were tested
against several bacterial strains (E. coli, P. aeruginosa, S. aureus, and
Bacillus subtilis) in order to assess their antibacterial capabilities,
with a wide range of concentrations (3.2e102 mg/mL) [24e31]. Even
at low concentration, a complete inactivation rate of the tested
bacterial cells was observed after incubation with the GO-Ag NPs
for 30 min. Similarly, the smaller Ag NPs modified GO sheets
showed more effective bactericidal ability compared to large Ag
NPs modified GO sheets [26]. The antibacterial activities of GO-Ag
NPs against both E. coli and B. subtilis were improved more than
one fold as compared to that of Ag NPs. Besides, this the Gram
positive bacteria displayed higher resistance than the Gram nega-
tive ones to the GO-Ag NPs [30].
Polymeric modified rGO was applied as a platform for Ag NPs to
enhance the stability of Ag NPs. Based on this strategy, several
polymers (poly diallyldimethyl ammonium chloride (PDDA), poly-
ethyleneimine (PEI), poly-dopamine (PDA) and poly acrylic acid
(PAA)) were applied to obtain such composites via different syn-
thesis routes (one-pot hydrothermal, in situ reduction and in situ
grafting) [32e35]. The average bactericidal activity of Ag NPs was
maintained in the polymeric rGO composites (100% inhibition),
with extended time of exposure (6 h) compared to the non-
polymeric Ag NPs-GO [33]. Moreover, the multifunctional GO-Ag
NPs nanocomposites were synthesized by integration with metal
oxides, e.g. titanium dioxide (TiO2) [36] and iron oxide [37], for new
applications. The rapid inhibition effect (67%) against E. coli in just
2 h evidently shows the synergistic influence of GO and Ag NPs on
the bactericidal features of the resultant nanocomposite particles.
On the other hand, metal oxides (MnO2, TiO2, Fe2O4 and ZnO)
were solely applied to hybridize with Gr-based materials in order to
enhance photocatalytic activity and synergistic antibacterial prop-
erties of the resultant composites [38e44]. Different techniques
were developed to homogenously decorate Gr-based materials
with metal oxide nanoparticles, viz. hydrothermal and solvo-
thermal, and the obtained hybrids were tested against several
bacterial species. The lowest bactericidal effect (82%) was obtained
with 100 mg/mL of Gr manganese ferrite (MnFe2O4) as compared to
Gr (37%) [44], while bacterial cells were totally inhibited upon the
application of GO-ZnO or GO-TiO2 nanocomposites [38,39].
Furthermore, the triple antibacterial effect of poly-L-lysine (PLL)-
rGO-CuNPs was evaluated against E. coli and S. aureus, and the re-
sults revealed that there is a long-term antibacterial effect (99%)
[45]. In addition, Gr-based materials were functionalized with
metals (Lanthanum (La), Ag and Cd) to increase their antibacterial
ability [46e48]. For instance, G-CdS metallic composites with ho-
mogenous distribution of CdS NPs on GO nanosheets displayed
higher bactericidal activity towards E. coli than that with the pure
CdS NPs.
2.2. Gr-based nanocomposite surfaces
Membranes may be one of the most significant marketable
products for Gr. Several industries, e.g. water desalination and gas
separation, could find application of Gr as a medium for different
membranes. Pristine or functionalized Gr membranes can have
outstanding potential in selective uptake and transportation of
ionic or molecular species. One of the first trials to assess the
antibacterial property of Gr-based surfaces was conducted by
Akhavan and Ghaderi (2009), where GO nanosheets were depos-
ited on TiO2 thin films and displayed 60% bacterial inhibition [49].
Another common route for the hybridized Gr membranes is the
polymer-GO, with various polymers (chitosan (CS) [50], poly vinyl
alcohol (PVA) [51], PLL [52,53], polyester (PE) [54], poly-
ethersulfone (PES) [55], polyamide (PA) [56], polypropylene (PP)
[57] and polyethylenimine (PEI) [58]) embedded via several tech-
niques, viz. layer by layer (LbL)-deposition, casting, hybrid grafting,
phase inversion and electrospinning. The antibacterial efficiency of
these polymeric Gr-based membranes ranged from 64 to 99%
[53,57]. The bactericidal activity of the membranes improved by
increasing the Gr content. These results demonstrate that the
antibacterial and antifouling properties can be enhanced by bind-
ing Gr materials to the membrane surface.
These promising results drove the research towards producing
more efficient polymeric Gr-based membranes with improved
antibacterial properties. In view of that, several Ag NPs - polymeric
Gr based membranes were studied. The GO-Ag NPs nanocomposite
was used to modify PES [59], cellulose acetate (CA) [60] or poly-
sulfone (PS) [61] membranes. The maximum antibacterial effi-
ciency of these membranes was 100%, due to the synergistic effect
of GO and Ag NPs.
Alternatively, the Gr-based composite coatings were produced
by deposition on metal surfaces, like Ti [62], stainless steel (SS) [63]
or copper (Cu) [64]. It was clear that the type of coated metal
significantly affected the antibacterial ability of the Gr-coated sur-
face. For example, when SS was coated with Gr, the biocidal activity
reached 95%, while this activity decreased to only 34% when Cu was
coated.
2.3. Gr-based hydrogels
The three-dimensional GO-based hydrogels have recently
attracted more attention due to their valuable features. Neverthe-
less, it is rather challenging to fabricate cheap, efficient and wide
spectrum bactericidal hydrogels that can be easily recycled.
Therefore, research was driven towards fabricating new Gr-based
composite hydrogels with high antibacterial capacity. One of the
early trials in this field was carried out by applying a simple and
eco-friendly technique to produce a multifunctional Gr-based
hydrogel employing a biocompatible agarose polysaccharide
which simultaneously functions as a physical cross-linking agent
and a stabilizer [65]. The newly fabricated hydrogel resulted in the
full reduction of bacterial growth. Moreover, some commercial
preservatives, e.g. benzalkonium bromide cationic surfactant, were
hybridized with GO to gain the dual antibacterial benefits of both
materials. The benzalkonium bromide/GO hydrogel displayed
strong bactericidal activities toward both Gram positive (91%) and
Gram negative (99%) bacteria [66]. The same “synergistic effect”
strategy was investigated by fabricating Ag/Gr hydrogels
 H.M. Hegab et al. / Carbon 105 (2016) 362e376 367

composites for enhanced levels of antibacterial efficacy. Several 3.1. Sheet size and concentration
hydrogels, like Ag/PVC/Gr [67] and Ag/Gr [68], were tested for their
antibacterial abilities, and the results revealed the complete The antibacterial activity of Gr-based materials was observed to
reduction of S. aureus and E. coli cellular growth. be influenced by the size of the GO sheet. Smaller sheet size
resulted in enhanced antibacterial activity for Gr-based surface
coatings. This concept was studied via investigating the simulating
2.4. Bacterial reduction of GO
effect of Gr sheets on bacterial cell membrane [79]. It was observed
that small Gr sheets rupture the phospholipid layer of the bacterial
One of the most confusing topics is the bacterial reduction of
membrane, while large Gr sheets stay flat on the top of this layer,
GO, where it contradicts the known bactericidal effect of Gr species.
where they destruct the membrane and turn over part of the
Some bacterial cells are capable of reducing GO nanomaterial by a
phospholipids. When the sheet area was decreased from 0.65 to
specific mechanism. This mechanism is determined by the direct or
0.01 mm2, the bactericidal activity of Gr surface coatings improved
indirect ability of bacterial cells to hydrolyze acidic groups com-
4-fold [80]. The greater antibacterial effect of smaller sheets is
bined with carbonic nanosheets in particular oxygen atoms. GO can
ascribed an oxidative stress mechanisms accompanied by the
act as a final electron acceptor in the bacterial respiration process
excessive defect density of these sheets. Moreover, GO sheet area
[69]. The first trial to bacterially reduce GO was conducted in 2010
hinders bacterial growth, in cell suspensions, by a cell wrapping
by using Shewanella with GO acting as a terminal electron acceptor,
mechanism. Under this environment, the bactericidal activity of GO
in which electron shuttling to GO is mediated by cytochromes.
is amplified as the sheet area increases, with evidence of complete
Subsequently, Shewanella was utilized for GO reduction in number
inhibition observed for sheets sized 0.65 mm2, after 3 h of incuba-
of studies by external electron shuttling intervened by the
tion. The same correlation was observed when the bactericidal
membrane-bound c-type heme cytochromes, and via self-
activity of GO sheets with various lateral sizes was assessed with
produced electron mediators [70,71].
E. coli cells [21]. GO sheets with greater lateral size showed a
Various types of bacteria were employed to assist in the
temporary powerful bactericidal activity compared to smaller ones.
reduction of GO. B. subtilis was applied to reduce GO and develop an
Nevertheless, cell wrapping was observed to be reversible, showing
enhanced supercapacitor [72]. The bacterial cells not only
that the noticeable effect of GO does not encompass bacterial in-
contribute towards the GO reduction, but also in producing the final
hibition and can be relatively designated as bacteriostatic [80]. The
composite through entrapment within the rGO. Extremophiles
size of Gr nanosheets is related to cellular uptake and several bio-
cells were also applied for GO reduction to produce an extremely
logical phenomena that rely on lateral dimension [81]. In general,
conductive Gr that can be employed in the manufacturing of mi-
the sharp edges, small size and rough surfaces of Gr nanosheets
croelectronics [73]. The extremophiles have the ability to reduce
facilitate their internalization into the bacterial cells compared to
GO through a particular mechanism that enables them to degrade
larger and smooth sheets [82]. The Gr nanosheets lateral sizes
toxic materials so as to survive under severe environmental con-
range from nano-GO (10 nm) to more than 20 mm, which is bigger
ditions. A different study was performed by Avinav et al. [74], in
than bacterial cells [83]. Additionally, lateral dimension and layer
which GO was produced from a cellulose precursor and was sub-
number have an impact on deformability, and also affect the spin
sequently reduced in situ by means of Gluconacetobacter xylinus. In
that can be employed by cellular forces, where small plates are less
two contradictory studies, E. coli was applied for the reduction of
deformable than large ones with the same layer number [84].
chemically exfoliated GO nanosheets [75,76]. In the first one, GO
Furthermore, the effect of Gr-based materials concentration on
nanosheets represent biocompatible spots for adsorption and
bactericidal activities was investigated in several studies. When GO
growth of the bacterial cells on their surfaces, whereas the resulted
suspensions, with different concentrations (5, 10, 20, 40 and 80 mg/
rGO nanosheets from the bacterial metabolism displayed inhibitory
mL), were exposed to E. coli cells, they resulted in the loss of bac-
effect for the bacterial growth on their surfaces [75]. However, the
terial viability as the GO concentration increased, until it reached
same bacterial cells have been used in the second study for GO
more than 90% with a GO concentration of 80 mg/mL [16,20,21].
reduction to Gr nanosheets that were dispersible in water [76].
Similarly, rGO dispersion with a concentration of 80 mg/mL eradi-
The bio-reduction of GO has several advantages in terms of
cates 76.8% of bacterial cells [16]. In addition, further higher con-
occurrence under ambient conditions in short time [77]. The
centrations (25e200 mg/mL) of Gr-based materials were studied in
bacterially-rGO showed outstanding electrochemical features [71],
terms of their effects on bactericidal activities [22]. P. aeruginosa
with the developed electroactive biofilm delivering 25-fold in-
gradually lost its viability with elevated concentrations of GO or
crease in the outward current in terms of electron release from
rGO, 75 and 100 mg/mL, respectively. These results suggested that
bacterial cells to electrodes [70]. The inward current was also
80 mg/mL is the threshold GO concentration that can lead to a
increased (74-fold), in terms of electron release from electrodes to
significant antibacterial activity (>90%), while this critical concen-
bacterial cells, by the developed biofilm compared to the naturally
tration is higher when rGO is used (100 mg/mL).
developed biofilms. Moreover, the bacterially-rGO has considerable
biocompatibility with fibroblast cells, even when applied at higher
3.2. Surface area
concentrations, e.g. 100 mg/ml. Hence, the green approach of the
bacterial reduction of GO confers a great applicable potential in the
Bacterial interactions of nanomaterials are greatly influenced by
biomedical field [78].
the nature of the surfaces [85]. Small NPs, less than 10 nm, usually
have a considerable portion of atomic surface exposure. Every atom
3. Gr-based materials features related to biocidal activity occurs on the surface in the case of monolayer Gr, in which each
atom is subjected to the contiguous medium on dual sides, leading
Gr-based nanomaterials vary in sheet size, surface area, rough- to increase in the speculative maximum surface area of the sp2-
ness, hydrophilicity, dispersibility and functionalization; and all of carbon sheet (2600 m2/g) [86]. This is superior to the surface area
these features can individually or interactively impact the interac- of other nanomaterials which have been investigated in microbial
tion of Gr with bacterial cells. The separate effect of each factor on systems. The GO single layer has a comparable area, however its
the viability of bacterial cells is illustrated in Fig. 3 and detailed in surface is controlled by roughness at atomic-scale. The number of
the following sections. layers in Gr nanosheets is vital as it determines bending stiffness
368 H.M. Hegab et al. / Carbon 105 (2016) 362e376

Fig. 3. The physiochemical properties of graphene nanomaterial relevant to its contradictory antibacterial effect. (A colour version of this figure can be viewed online.)

and specific surface area [87]. The adsorptive capacity for biotic contact angle 90 ). Normally, bacterial cells have higher affinity for
molecules is assumed to significantly increase as layer number hydrophobic surfaces due to hydrophobic interactions, which de-
decreases. pends on the type of bacteria tested [91]. Therefore, hydrophilic
surface modification is an effective approach for the establishment
of bactericidal surfaces by using polymeric materials, e.g. highly
3.3. Surface roughness
hydrated polyethylene glycol (PEG). However, some polymeric
chains can dehydrate at high ionic strengths, resulting in a lowering
The physical structure of the surface is a crucial factor affecting
of the efficiency for preventing bacterial adhesion on such surfaces
interaction with bacterial cells. Increased surface roughness im-
[95]. It is generally considered that the fast adsorption taking place
proves bacterial cell adhesion. Both high surface area and deep
on hydrophobic surfaces is accompanied by strong binding forces,
terrains in the surface result in greater colonization [88]. The sur-
in contrast to hydrophilic surface adhesion which follows the
face morphology features include peaks and valleys in the mem-
proposed model of reversible/irreversible adhesion [96]. A vulner-
brane surface resulting from the fabrication process [89]. Therefore
able and reversible initial bacterial adhesion stage was observed,
there is a difference between bacteriostatic surfaces that inhibit
split-up at distances of several nanometers, which could be de-
attachment and bactericidal surfaces that efficiently eradicate
tached by shear forces or spontaneous desorption. Subsequently,
bacterial cells on contact [90], although both surfaces are usually
production of extracellular biopolymers or stabilization into exist-
categorized as antibacterial. Some functionalities displayed
ing polymers results in the conversion of this adhesion to be irre-
powerful antibacterial action by generating membrane perme-
versible. The adsorbed water is replaced by these bridging
ability, due to the surface morphology, which can be employed to
polymers (separation spaces of <1 nm) along with the neutraliza-
destroy bacterial cells. Normally, such functionalized surfaces have
tion of the electrostatic repulsion [91]. Bacterial surface adhesion in
considerable roughness or ridges with high frequency, which can
aquatic environments typically encompasses hydrophobic groups
cause cracking of bacterial cell walls during contact [90]. Although
in the cell walls (nonpolar surface proteins), in which they help
the Gr sheet edges follow the latter mechanism, their basal planes
cells to make contact with the substratum, providing alterations in
are believed to be smooth at the atomic level. Thus, the interaction
surface polymers. These results in different functional groups
of the bacterial cells with Gr is expected to be greatly dependent on
approaching the surface for the creation of polymeric interactions
the alignment of the surface in relation to the bacteria [91].
[89]. Commonly, the adhesion ability of hydrophobic bacteria on
hydrophobic surfaces is greater than that of hydrophilic bacterial
3.4. Hydrophilicity cells [53,97].

The adhesion of bacterial cells on any substrate surface and their

ability to accumulate biofilm on it are influenced by its chemistry. 3.5. Dispersibility
GO sheets have amphiphilic nature with partial hydrophobic sur-
faces with some hydrophilic spots (contact angle 40e50 ) [92e94], The effective size of spherical NPs increases whenever they
while the Gr sheets have a hydrophobic surface (approximately aggregate in certain media, however, they can maintain the
 H.M. Hegab et al. / Carbon 105 (2016) 362e376
majority of their surface area [98]. They are able to attach via any
contact point throughout sphere to sphere aggregation, except in
the case of dissolution and reprecipitation of NPs, which may lead
to partial fusion [99]. In contrast, sheets usually stack face to face,
which contributes to the loss of part of the surface area upon
irreversible aggregation during filtration or centrifugation pro-
cesses. Regarding the GO dispersion, it comprises two-dimensional
carbon sheets [16], which are stable due to the great number of
different functional groups with hydrophilic nature, e.g. hydroxyl,
carboxyl, and epoxy groups [100]. This is one reason why GO has a
higher bactericidal activity compared with rGO. The rGO sheets are
formed by the reduction of GO nanosheets, and the resultant size is
approximately nine times bigger than that of GO nanosheets.
Therefore they easily aggregate, leading to a lower antibacterial
activity of rGO particles. Dynamic simulation was applied to
investigate the interaction of Gr nanosheets with a lipid bilayer in
cell membrane, in terms of Gr thicknesses, e.g. single versus multi-
layered Gr [101]. It was found that single and few-layered Gr
nanosheets can unexpectedly penetrate the cellular bilayer and
orient themselves in parallel within the mid of the bilayer.
3.6. Functionalization
Adhesion for some bacterial species may be related to surface
chemistry [102], in which a surface charge is required owing to
ionization of the acidic/basic functional groups in their cell wall
[103]. Commonly, the bacterial cell wall has a negative charge in
aqueous suspensions with neutral pH. Nevertheless, the level of the
charge differs from one species to another and is affected by several
factors, viz. the culture age, nutritional medium, ionic strength, cell
surface structure and pH. The increased electronegativity of the cell
wall causes the reduction of cellular adhesion on to the surface.
Accordingly, several treatment or surface modification methods
have recently been developed for the fabrication of altered bacte-
ricidal charged surfaces. These techniques involve functionalization
[53,97,104e106], derivatization [107] and surface polymerization
[108,109], in which chemicals are used to change the surface fea-
tures. The partially hydrophobic nature of GO surfaces along with
some hydrophilic regions [92e94] permit these surfaces to be
engaged in metal ion complexing and hydrogen bonding [110].
Moreover, GO sheets contain negatively charged edges accompa-
nied with carboxylate groups [111]. In contrast, only the defect sites
or edges of the hydrophobic Gr surface are able to biochemically
react. rGO has an intermediary hydrophilic nature and reactivity, as
it contains basic defective spots due to oxygen removal during its
production [112].
4. Antibacterial mechanisms of Gr-based materials
4.1. General mechanisms
Bacterial cells could be eventually destroyed over time, when
they come into contact with Gr-based materials, due to several
mechanisms, viz. membrane stress, oxidative stress, and/or wrap-
ping isolation. These mechanisms can act separately or together to
either bactericidally or bacteriostatically inhibit bacterial growth.
The three common antibacterial mechanisms of Gr-based materials
are schematized in Fig. 4 and elucidated in the following sections.
4.1.1. Bacterial membrane stress
Cellular injury might occur from the impact of physical mem-
brane disruption that has been detected as the biological effects of
CNTs and fullerene [20]. Most E. coli cells lose cellular integrity and
tend to be flattened after incubation with rGO or GO dispersions
[16]. This demonstrates that bacterial cells can be irreversibly
369
destroyed after a direct exposure to Gr-based materials [22].
A novel explanation for this antibacterial mechanism has been
discovered. It revolves around the damage caused by the extraction
of phospholipids from the E. coli cell membrane (Fig. 4) [113]. This
extraction was monitored by transmission electron microscope
(TEM) after the exposure of E. coli cells to GO nanosheets. The
bacterial cells were initially tolerant to GO for a short time, after
which a partial loss of cell membranes integrity occurred, with
some cells showing a lower density of surface phospholipid. Finally,
the cell membranes were exposed to severe damage and some
completely losing their cytoplasm (Fig. 5A, B) [113]. This sequence
of destruction was confirmed by a molecular dynamic simulation,
illustrating more details about the damaging mechanism. The Gr
nanosheet starts the swing mode, in which it vibrates around the
atom for a time of ten to a hundred nanoseconds. This is followed
by the insertion mode, where the sheet edge moves in and pierces
the cell membranes as a result of the strong van der Waals in-
teractions with the membrane lipids and hydrophobic interface.
Finally, the nanosheets strongly extract the phospholipids from the
lipid bilayers located on the cell surface during the extraction step.
These observations confirm that the insertion and lipid extrac-
tion destructive mechanisms of Gr-based materials result in crucial
membrane stress, leading to a reduction in cell viability [113]. The
peak perturbation of the lipid bilayer by Gr sheets was observed for
unoxidized and large nanosheets, in which the lipid bilayer is
penetrated through the contact with the Gr sheet edges. These
atomically thin sharp edges of the Gr nanosheets spontaneously
pierce the membrane, resulting in the reduction of the energy
barrier required for membrane penetration. The negative charges of
the bacterial cells also play an important role in their interaction
with the sheet edges which act as good electron acceptors, and
therefore contribute toward bacterial cell membrane damage. In
contrast, the hydrophilic nature of GO sheets result in them mostly
attaching to the membrane surface without piercing the lipid
bilayer [80]. The level of the cell membrane destruction by rGO is
higher than that caused by the GO nanosheets, due to the abun-
dance of sharp edges along with better charge transfer in the rGO
nanosheets leading to further destruction of the bacterial cell
membrane [63].
Furthermore, the effect of Gr nanosheets on bacterial cell
membrane damage was studied by measuring the efflux of the
intracellular components of the bacterial cells upon exposure to the
nanosheets [63]. This was achieved by quantifying the RNA con-
centration in the medium. It was found to be high when the bac-
terial cells were exposed to GO or rGO compared to the control
sample. The interaction of the bacterial cells with the sharp edges of
the nanosheets resulted in higher destruction of the cell membrane
of the Gram-positive bacteria, deficient in the external membrane,
compared to the Gram-negative ones having it [22]. Some bacterial
cells, e.g. P. aeruginosa, comprise of four layers of shielding mem-
branes [114], with an external membrane of a surface strongly
cross-linked lipopolysaccharide [115]. This leads to low absorbency
of hydrophilic solutes, causing the resistance of the bacterial cells to
several bactericidal agents.
In the case of the stable suspension of Gr-based nano-
composites, e.g. GO-Ag, GO sheets interact with bacterial cells, for
instance, the resulting bactericidal activity of the GO-Ag nano-
composites is observed via the interaction between bacteria and Ag
NPs supported on GO sheets. The bactericidal effect of Ag-GO
nanocomposites appears to be initiated by the “capturing-killing”
mechanism that plays a role in the deposition of bacterial cells and
increases their contact with the fabricated Ag NPs [116].
4.1.2. Oxidative stress
There are additional mechanisms related to the physicochemical
370 H.M. Hegab et al. / Carbon 105 (2016) 362e376

Fig. 4. Permanent and temporary bacterial cell growth inhibition mechanisms by Gr-based materials. (A colour version of this figure can be viewed online.)

Fig. 5. GO nanomaterial cellular destructive effect against E. coli cells; (A): TEM images of untreated cells, (B): Cells treated with GO nanosheets, (C): DNA fragmentation of cells
exposed to GO, rGO and Ag NPs. Reprinted with permission from Refs. [20,138].

interactions of Gr-based materials with bacteria which may have a
significant role in their antibacterial activity. The key role of
oxidative stress has been proposed in the antibacterial activity of
GO. The action of oxidative stress on bacteria results in the oxida-
tion of lipids, nucleic acid and proteins which can eventually lead to
cell membrane destruction and cellular growth inhibition [80]. This
inhibition occurs via ROS generation, which might change the
cellular redox condition. Oxidative stress as well as ROS production
were observed to be some of the main mechanisms in cellular
defense when particles are absorbed. The oxidative stress could be
 H.M. Hegab et al. / Carbon 105 (2016) 362e376
accelerated by NPs through the disturbance of the balance between
the intracellular oxidant and antioxidant processes [117,118],
occurring as a result of the disturbance of bacterial antioxidant
defense reactions (ROS-independent oxidative stress) [16,119]. The
ROS usually includes the hydroxyl radical (OH), superoxide radical
(O2) and hydrogen peroxide (H2O2), that destroy cellular compo-
nents, particularly proteins and DNA [119,120]. The ROS damaging
effect was confirmed by the irreversible DNA laddering occurring
upon the exposure of bacterial cells to GO (24 h) (Fig. 5C), while rGO
did not display the same effect [22]. This proposes that bacterial
cells need longer exposure time to rGO in order for DNA to be
fragmented or otherwise the cell death mechanism by rGO could be
unlike GO. This was further verified by a PDA-based experimental
procedure, in which an atomic force microscope (AFM) probe was
functionalized with GO nanosheets, and employed to screen the
interaction between the bacterial cell membranes and GO [121].
The results showed that this interaction is mainly repulsive, with
only infrequent attachment forces being monitored during probe
pull-off, which was attributed to lipopolysaccharide bridging.
The oxidative capacity of smaller GO sheets increase due to their
greater defect densities. Lipid peroxidation is thought to be the
principal oxidative pathway resulting in the development of
oxidative stress in bacteria incubated with GO, owing to the adja-
cent contact between GO nanosheets and bacterial cell membranes
[122]. Lipid peroxidation takes place as a chain reaction that is
started by the lipid molecules oxidation via ROS (Fig. 4), resulting in
the formation of lipid peroxide radicals which initiate the cell
membrane oxidative destruction [123].
4.1.3. Wrapping isolation
Bacterial cells can be biologically isolated from their growth
medium when Gr sheets enclose them [124], and therefore they
cannot consume nutrients to grow, as permeability via the cell
membrane is blocked, leading to cell death [21]. GO nanosheet
layers can also quarantine bacterial cells, even though they may
have greater permeability compared to Gr sheets (Fig. 4). It was
revealed that the majority of E. coli cells were separately wrapped
with GO nanosheets small layers, while they were typically
entrapped in aggregates of large rGO sheets [16]. Nevertheless, cell
inhibition by GO embedding was reversible and all the originally
viable cells may possibly be recovered by their separation from GO
nanosheets via several techniques, e.g. sonication [80]. A molecular
Fig. 6. Schematic diagram illustrating the antibacterial strength of different Gr-based suspensions. (A colour version of this figure can be viewed online.)
371
biological simulation has verified that small Gr sheets
(~5.9
6.2 nm2) can be locked in bacterial cell membranes
comprising phospholipid components [125], with the possibility of
the some sheet internalization by bacterial cells. The different ac-
tivities of rGO and GO bring up the important role of Gr-based
materials dispersion/aggregation in their bactericidal effects [16].
4.2. Gr-based suspension/membrane antibacterial capabilities
When GO nanosheets are immobilized on a coated surface, their
interaction with bacteria may be considerably different than that in
suspension form. The alterations in the GO nanosheets physico-
chemical features, e.g. sheet size, also may have a diverse effect
when employed on a surface [80]. This divergence proposes that
the antibacterial effect of GO includes different mechanisms in the
suspension phase compared to a GO-based coated surface. The
inactivation of cell growth by huge nanosheets of GO suspension is
reversible, which elucidates that the GO suspensions/bacterial cell
interaction will not lead to cell growth inhibition as noticed for a
GO-based surface coating [80]. The reason behind this is the high
number of viable cells interacting with GO nanosheets in the bac-
terial cell-GO aggregates. The effect of aggregation form on this
interaction is widely suggested to be orientation reliant [126e129].
Cellular membrane destruction initiated by phospholipid drawing
out or nanosheet piercing by GO is primarily caused by orthogonal
interaction with GO nanosheet edges [127,128]. Alternatively, con-
tact with basal planes is required, through a quite anonymous
mechanism, for the GO antibacterial activity [126,129]. GO-coated
surfaces lessen orthogonal cell interaction with nanosheet edges
and improve contact with basal planes owing to flat sheet
assembling.
This was further confirmed by studies on the effect of the
availability of the basal planes, compared to sheet edges, on the
GO antibacterial features [126,129]. When the sheets are entirely
flat on the surface and cells slightly interact with the sheet edges,
GO nanosheets were still observed to inhibit bacteria that
approach the surface [129]. These conclusions suggested that the
interaction with the sheet edges, followed by direct penetration of
the cell membrane by GO nanosheets, is not necessary for the
antibacterial effect of GO. Furthermore, the direct interaction be-
tween GO and bacterial cells is required for efficient bactericidal
activity, as no effect on cell viability was detected for planktonic
372 H.M. Hegab et al. / Carbon 105 (2016) 362e376

growth not in direct contact with GO nanosheets, compared to
bacterial cells adhered to all GO surfaces [16,130]. Briefly, bacterial
cell toxicity differs according to the orientation of the sheets
assembled on the surface. The sharp edges of rGO and GO nano-
sheets that are exposed to interaction with the bacterial cells
induce membrane rupture, while the interfacial basal planes at the
surface showed no bactericidal activity. The orientation of the rGO
or GO sheets in suspension may facilitate or impede the contact
with bacterial cells in a way of edgewise interaction. From Table 1
the biocidal strength order of Gr-based nanomaterials dispersions
(Fig. 6) could be considered as follows: Gr hybrid NPs, GO and Gr.
While the antibacterial strength of Gr-based nanomaterial surfaces
(Fig. 7) could be summarized in the order of Gr hybrid NPs, Gr and
GO.

Fig. 7. Schematic diagram illustrating the antibacterial strength of different Gr-based surfaces. (A colour version of this figure can be viewed online.)
 H.M. Hegab et al. / Carbon 105 (2016) 362e376
5. Potential of Gr antiviral ability
Despite the availability of attenuated and inactivated vaccines
for some viral strains, e.g. pseudorabies virus (PRV) and porcine
epidemic diarrhea virus (PEDV), these viral infections are still often
detected, due to the development of new modified strains. The
absence of effective specific antiviral therapy for such viral diseases,
highlights the urgent need to develop new antimicrobial agents.
The antimicrobial ability of Gr material derivatives has been
extended to cover viruses due to their great surface area and their
exceptional physical and chemical features, with less debatable
mode of action compared to that in bacteria [131,132]. The obvi-
ously low toxicity allowed Gr derivatives to be potential candidates
for the future generation of antimicrobial compounds.
Researchers had been interested in the antiviral ability of Gr
derivatives, following different approaches, since 2012. A recent
trial was performed by Sametband et al. (2014) who observed that
partially reduced sulfonated GO (rGO-SO3) and GO could deactivate
herpes simplex virus (HSV-1) infection via cellular attachment at
small concentrations [133]. Both GO materials carry several
negative-charged groups, e.g. sulfonate and carboxyl, which are
identical to heparan sulfate (HS) occurring on the cell surface,
resulting in the competition with the HS to bind HSV-1. Another
antiviral strategy was applied by Akhavan et al. (2012) in which Gr-
tungsten oxide composite thin films were applied in the photo-
inhibition of viruses under visible light irradiation [134]. Simi-
larly, Hu et al. (2012) employed a new photocatalyst, namely GO
functioned aptamer, in which this photocatalyst was synthesized
using the target recognition aptamer and the photosensitive GO.
This photocatalyst efficiently inhibited viruses, and destructed their
proteins capsid along with their nucleic acids under irradiation.
This resulted in the carbonylation of the proteins and oxidation of
the nucleic acid bases, particularly guanosine, along with the
reduction of GO [135]. Moreover, a GO-based material for the
intracellular transport of deoxyribozyme (Dz) was reported, in
which it targets the mRNA of hepatitis C virus (HCV) to block the
replication of HCV gene [136].
The effective antiviral ability of both rGO and GO is due to the
distinctive single-layer configuration and negative charge,
compared to Gt and GtO which showed lesser antiviral activity,
concluding that the nanosheet structure is vital for antiviral ability.
Moreover, GO inhibited viruses by mechanical destruction before
entering the viral structure. Nevertheless, the potential viral
deactivation effects of Gr derivatives on different types of viruses
have not been studied, along with their mode of action by which
they influence viruses [137].
6. Concluding remarks
The strong antibacterial activity of Gr-based materials against a
wide range of bacterial cells, including Gram negative and positive
strains, were demonstrated. These antibacterial properties are
believed to be caused by chemical and physical interactions upon
the direct contact of sheets with bacteria, in which the bacterial cell
membrane seems to be the main target of this activity. The resul-
tant bacterial membrane destruction was recognized by morpho-
logical alterations in the cell structure, RNA leakage, release of
intracellular components and variations in the transmembrane
capacity. This damage is attributed to the atomic-based Gr sharp
edges, which could pierce the bacterial cell membrane leading to a
physical disruption in its integrity. Cell membrane destruction
could also be chemically mediated by lipid peroxidation initiated by
the oxidative ability of GO. On the other hand, in spite of the sci-
entific awareness for the importance of Gr-based materials
employment in antibacterial surfaces, there is a limited knowledge
373
and inconsistent interpretations of the required Gr material fea-
tures for efficient antibacterial activity. The contradictory expla-
nations regarding the bactericidal properties of Gr-based materials
can be ascribed to the variations in the physicochemical features for
each individual sample. The structural chemical and physical
characteristics, viz. nanosheets thickness and size, dispersibility
and oxidation capacity, are distinctive, depending upon the Gt
source and the preparation method (oxidation/exfoliation). Thus,
an accurate and comprehensive evaluation with regard to these
factors is essential for prospective studies on the bactericidal
properties of Gr-based materials. Moreover, the inconsistent
bactericidal outcomes of the suspension form and coated surfaces
highlight the need for a precise assessment of the antibacterial
activity of the different forms of Gr-based materials.
Acknowledgments
Hanaa Hegab appreciatively acknowledges the supportive
funding from the Australian government and University of South
Australia through an IPRS scholarship. Additionally, the authors
would like to acknowledge the support of Water Research Australia.
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