Advances in Colloid and Interface Science 236 (2016) 101–112

Contents lists available at ScienceDirect

Advances in Colloid and Interface Science

journal homepage: www.elsevier.com/locate/cis

Historical perspective

Antimicrobial graphene family materials: Progress, advances, hopes

and fears
Anna Lukowiak a,⁎, Anna Kedziora b, Wieslaw Strek a
a
Institute of Low Temperature and Structure Research, Polish Academy of Sciences, 2 Okolna St., 50-422 Wroclaw, Poland
b
Institute of Genetics and Microbiology, Faculty of Natural Sciences, University of Wroclaw, 63/77 Przybyszewskiego St., 51-148 Wroclaw, Poland

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

Available online 20 August 2016 Graphene-based materials have become very popular bionanotechnological instruments in the last few years.
Since 2010, the graphene family materials have been recognized as worthy of attention due to its antimicrobial
Keywords: properties. Functionalization of graphene (or rather graphene oxide) surface creates the possibilities to obtain ef-
Graphene family ficient antimicrobial agents. In this review, progress and advances in this field in the last few years are described
Graphene oxide and discussed. Special attention is devoted to materials based on graphene oxide in which specifically selected
Functionalization
components significantly modify biological activity of this carbon structure. Short introduction concerns the
Composite
Biological activity
physicochemical properties of the graphene family materials. In the section on antimicrobial properties, pro-
posed mechanisms of activity against microorganisms are given showing enhanced action of nanocomposites
also under light irradiation (photoinduced activity). Another important feature, i.e. toxicity against eukaryotic
cells, is presented with up-to-date data. Taking into account all the information on the properties of the described
materials and usefulness of the graphene family as antimicrobial agents, hopes and fears concerning their appli-
cation are discussed. Finally, some examples of promising usage in medicine and other fields, e.g. in phytobiology
and water remediation, are shown.
© 2016 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
2. Graphene oxide and other graphene family nanomaterials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
2.1. Short characterization of graphene materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
2.2. Antimicrobial properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
3. Progress and advances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
3.1. Recent reports on antimicrobial activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
3.2. GO functionalization and nanocomposites with graphene family materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
3.3. Antimicrobial properties enhanced by photoreactivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
4. Hopes and fears . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
4.1. Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
4.2. Promising applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
4.3. Invention disclosure related to antimicrobial graphene-based materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109

Abbreviations: ESBL, extended spectrum β-lactam; ESKAPE, a group of the most frequent nosocomial pathogens Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae,
Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.; G−, Gram-negative bacteria; G+, Gram-positive bacteria; CFU, colony forming unit; CNSs, carbon nanoscrolls;
EDTA, ethylenediaminetetraacetic acid; GO, graphene oxide; GrO, graphite oxide; GONMs, graphene oxide-based nanomeshes; GQDs, graphene quantum dots; MDR, multidrug resistant;
MEF, mouse embryo fibroblast; MIC, minimal inhibitory concentration; MIC50, concentration that inhibits 50% of bacterial isolates; MRSA, methicillin-resistant Staphylococcus aureus; NIR,
near infrared; NPs, nanoparticles; Pc, phthalocyanine; PEG, polyethylene glycol; rGO, reduced graphene oxide; rGrO, reduced graphite oxide; ROS, reactive oxygen species; SWCNT, single-
walled carbon nanotube; MWCNT, multi-walled carbon nanotube; Vis, visible; WHO, World Health Organization.
⁎ Corresponding author.
E-mail address: A.Lukowiak@int.pan.wroc.pl (A. Lukowiak).

http://dx.doi.org/10.1016/j.cis.2016.08.002
0001-8686/© 2016 Elsevier B.V. All rights reserved.
102 A. Lukowiak et al. / Advances in Colloid and Interface Science 236 (2016) 101–112
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
Multidrug resistant (MDR) pathogens are a significant problem in
infections worldwide. In 2009, the Infectious Diseases Society of
America highlighted ‘ESKAPE’ pathogens. Six of the pathogens, the
most frequently found in hospitals and the most difficult to treat,
were included in this group: two of the Gram-positive bacteria
(G+: Enterococcus faecium and Staphylococcus aureus) and four of
the Gram-negative species (G−: Klebsiella pneumoniae, Acinetobacter
baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) [1]. Mul-
tidrug resistance among pathogens is one of the major problems in
therapeutics. The MDR group of pathogens (not related to ‘ESKAPE’
and nosocomial infections) cannot be treated with typical antimicro-
bials. These pathogens are resistant to most of the known and used
antibiotics (e.g. Escherichia coli as MDR to carbapenems and cephalo-
sporins [2]). As indicated in the report from Southern Asia [3], the
most popular MDR bacteria strains from wounds of the hospitalized
patients are 5 of the ‘ESKAPE’ pathogens: methicillin-resistant
S. aureus (MRSA) in the G+ group and extended spectrum β-lactam
resistant Enterobacteriaceae in the G− group (ESBL: K. pneumoniae,
A. baumannii, P. aeruginosa, Enterobacter cloacae, and Proteus mirabilis).
According to WHO (data from 2015), mortality among patients infected
with MDR pathogens is two-fold higher than among those infected with
drug-sensitive strains of bacteria.
Antibiotics have been known for decades and their overuse and irra-
tional application are causing increasing multidrug resistance in bacte-
ria and fungi. Therefore, there is a strong need to find alternative ways
for killing pathogens. Local and global events or programs, such as the
National Program for Antibiotics Protection in Poland or the European
Antibiotics Awareness Day on November 18th and World Antibiotic
Awareness Week, have been established to draw attention to this
problem.
Fortunately, due to the development of nanobiotechnology and
novel bioactive materials, we can put into practice more and more
ideas and solutions. One of the new possibilities in the research area
that have appeared in the last few years is based on the graphene family
materials that might be found in diagnostics, therapeutics or industry as
(1) antibacterial killing factors, (2) drug and gene delivery systems,
(3) bioimaging and photothermal therapy agents, (4) materials for tis-
sue engineering, and (5) biosensors [4–7]. Due to their bactericidal
and cell anti-adhesive properties, they are promising materials for
other applications, such as components for food packaging foils and
additives to drugs, textiles or medical and dentistry equipment.
A few reviews on biologically active carbon-based materials have
already been published [4,5,8–14]. Using this information, as well as re-
cently published data and our experience in graphene oxide and anti-
bacterial nanomaterials, we would like to briefly present antimicrobial
properties of the graphene family materials and to draw attention to
the promising applications of these structures and their functionalized
derivatives, and to the worries related to their usage.
2. Graphene oxide and other graphene family nanomaterials
2.1. Short characterization of graphene materials
Graphene, a layer of single-atom-thick carbon atoms closely packed
into a honeycomb two-dimensional lattice, is the main representative of
the carbon-based family nanomaterials. Other 2D structures which be-
long to this group are few-layer-graphene, ultrathin graphite, graphene
oxide, graphite oxide, and reduced graphene oxide, as well as carbon
nanotubes. They have been hailed as materials of the future due to
110
110
their unique thermal, mechanical, electrical or optical properties. Bio-
logical studies have recently shown that they are also promising candi-
dates for different medical applications, e.g. as antimicrobial agents.
The properties of graphene are a result of the sp2 hybrid carbon
framework, whereas in graphene oxide (GO) and graphite oxide
(GrO), a large fraction (0.5–0.6) of carbon is sp3 hybridized and cova-
lently bonded with oxygen in the form of epoxy (C\\O\\C) and hydrox-
yl (C\\OH) groups. The remaining carbon is sp2 hybridized and bonded
either with neighboring carbon atoms or with oxygen in the form of car-
boxyl (\\COOH) and carbonyl (C_O) groups which predominantly
decorate the edges of graphene sheets. GO is therefore a 2D network
of small sp2 carbon domains in a sp3-bonded matrix.
GrO can be easily obtained from graphite flakes at high yield under
oxidizing conditions and three main synthesis routes can be pointed
out. The modified Hummers method [15,16] is the most conventional
way, where a strong oxidant KMnO4 is used for graphite oxidation in
the presence of H2SO4, NaNO3, and H2O2. The second method is based
on the work performed by Brodie [17] who investigated the reactivity
of flake graphite by adding potassium chlorate to slurry of graphite in
fuming nitric acid. A similar procedure, but with adding chlorate in mul-
tiple aliquots over the course of the reaction (in the presence of concen-
trated H2SO4 to increase the acidity of the mixture), was used by
Staudenmaier [18]. Importantly, it has been demonstrated that the
products of these reactions show strong variance, depending not only
on the particular oxidants used, but also on the graphite source (natural
or synthetic graphite flakes) and reaction conditions. Different lateral
sizes (from several nanometers up to several micrometers) or the com-
position of GO structures affects physical, chemical, optical, and electri-
cal properties of the nanosheets.
Oxidized graphite usually retains its stacked structure (Fig. 1a).
Chemically, the two oxides – graphite oxide and graphene oxide – are
similar, but GO flakes are usually one or a few layers thick. Graphite
oxide can be exfoliated to graphene oxide (Fig. 1b) by thermal, mechan-
ical or sonochemical methods [19,20]. In this review, we have tried to
use the correct term – GrO or GO – to name the described materials.
However, in the literature authors sometimes do not specify which
oxide forms they have in mind and in this case, we decided to use the
GO definition.
The surface oxygen functionality of GO and GrO ensures its hydro-
philicity, so the flakes can be easily dissolved (at low concentration)
or dispersed in water or other polar solvents. Thus, the oxides can be
successfully used to be further functionalized, to form nanocomposites
or can be uniformly deposited from water-based solutions as thin
films on different substrates. The epoxy, hydroxyl, and carbonyl groups
of platelets as well as double bonds on the GO and GrO sheets are
chemically active. An ideal approach to the chemical modification
of graphene (graphite) oxide would utilize reactions of these groups
[19,21]. GO (and other graphene-based materials) can also exhibit
non-covalent binding via π–π or σ–π stacking and van der Waals inter-
action [22–24]. The non-covalent approach offers a non-destructive
way to modify the surfaces of carbon-based materials. A number of sur-
face modifiers including aromatic compounds, small-molecular surfac-
tants, amphiphilic polymers, and biomacromolecules have already
been described [25,26]. These features of GO and GrO – different than
in the case of graphene – make this material particularly interesting.
Therefore, we would like to focus mainly on this carbon material.
2.2. Antimicrobial properties
The mechanism of antibacterial activity of graphene materials is
still not well explained. In the literature, few possibilities have been
 A. Lukowiak et al. / Advances in Colloid and Interface Science 236 (2016) 101–112
Fig. 1. TEM images of graphite oxide (a), graphene oxide (b), and Ag-doped graphite oxide (c).
described and the proposed mode of GO action and effect of its interac-
tion with bacteria are presented in Table 1. Detailed reports on the re-
search can be found in published reviews (e.g. [5] and [9]). Here, we
give just some examples to show a general overview of the microbial
toxicity mechanisms.
One of the most interesting and the simplest modes of action is the
mechanical damage of bacterium cell envelope by some carbon forms.
According to Park et al. [27] the paper composed of reduced graphene
oxide (rGO) exhibited bacterial (B. cereus) attachment and subsequent
bacterial growth showing no cytotoxicity also to different mammalian
cell lines. However, in contrast, Hu et al. [28] have shown that the GO
and rGO paper can effectively inhibit the growth of E. coli bacteria
while showing minimal cytotoxicity. In the case of GO nanosheets de-
posited on stainless steel, it was found that the cell damage of the bac-
teria (E. coli or S. aureus) caused by direct contact of the bacteria with
extremely sharp edges of the nanowalls was an effective mechanism
in bacterial inactivation [29]. Moreover, the GO nanowalls reduced by
hydrazine were more toxic to the bacteria than the unreduced GO,
and G+ bacteria were more sensitive to these materials, although in
other studies rGO particles in suspension were observed to be signifi-
cantly more toxic to G− bacteria [30]. The aforementioned first studies
showed that the plates' edges have a significant role in the mechanical
damage of bacteria cells and when the graphene-based surface was
smooth, no antibacterial effect was observed. Moreover, it was proved
that interaction of GO with bacteria depends not only on the physico-
chemical properties of GO (size, shape, oxidation degree, etc.) but also
on the facilities of the microorganism cell — the individual features of
bacteria (build structures, presence or lack of any structures such as bac-
teria cell envelope, age of bacteria cell, stress condition in the environ-
ment, and metabolism) have an important influence on bacterial
sensitivity to carbon materials.
A similar activity through mechanical interaction was earlier dem-
onstrated for single-walled carbon nanotubes (SWCNTs). The cell mem-
brane damage resulting from direct contact with SWCNT was shown as
a likely mechanism leading to bacterial cell death [31]. The mechanical
damage was confirmed by a molecular-level study of DNA microarrays,
the analysis of gene expression, and SEM observation of E. coli cells after
incubation with carbon nanotubes [32]. Bacterial cell damage and the
alteration of gene expression were much more pronounced in results
from SWCNTs than in those from multiwalled carbon nanotubes
(MWCNTs). As suggested by the authors, the enhanced bacterial toxicity
Table 1
Mechanisms and effects of graphene oxide (GO) interaction with Gram-positive (G+) and/or Gram-negative (G−) bacterium cell.
GO interaction with bacteria
Proposed mechanism Effect
Physical contact interaction — entrapment of bacteria Cell death
Cell membrane damage, efflux of cytoplasmic materials, Cell death
decreasing metabolism
ROS production, glutathione loss, oxidative stress Cell death
Adhesion of the bacteria cell to the graphene surface, Cell growth, GO reduction
proliferation and stimulation of the biofilm formation
103
of SWCNTs may be attributed to (1) a smaller nanotube diameter that fa-
cilitates the partitioning and partial penetration of nanotubes into the cell
wall, (2) a larger surface area for contact and interaction with the cell sur-
face, and/or (3) unique chemical and electronic properties conveying
greater chemical reactivity. Individually dispersed SWCNTs, that were
more toxic towards bacteria, both G− (E. coli and P. aeruginosa) and G+
(S. aureus and B. subtilis), than SWCNT aggregates, were visualized as nu-
merous, sharp, moving “nano darts” which hit and damage bacteria cells
[33]. The authors also suggested that inhibiting cell growth, oxidative
stress or Co metal residues were not the major causes responsible for
the death of cells in the latter case.
Other, more complex, mechanisms of action have been demonstrat-
ed for graphene-based structures as well. Except aforementioned
disrupting the integrity of the cell membrane (mechanical damage of
the cell), these materials can (1) wrap around the bacteria isolating
them from the environment, (2) generate harmful reactive oxygen spe-
cies (ROS), (3) extract phospholipid molecules of the bacteria by the
presence of the lipophilic graphene, and (4) lower the metabolic activity
of the bacterial cells [34–38]. In the case of GO, ROS production is often
mentioned as the major factor killing bacteria (indifferent for human
and animal cells).
Nano-GO has been reported to be biocompatible as well, and the fa-
vorable cell growth on GO film has also been observed. Akhavan and
Ghaderi [39] showed that E. coli cells cause reduction of graphene
oxide (deoxygenation by 60%) in the metabolic process. It was found
that the GO sheets could act as biocompatible sites for adsorption and
proliferation of the bacteria. On the other hand, bacteria cells main-
tained sensitivity to rGO which inhibited the proliferation of E. coli rods.
There are not many examples of the antifungal mechanism of the
graphene family materials in the literature. According to Sawangphruk
et al. [40] inhibition of the mycelia growth results probably from direct
contact of rGO nanosheets with the cell wall (Aspergillus niger, Aspergil-
lus oryzae, and Fusarium oxysporum). The authors suggest that there is a
chemical reaction between oxygen (from rGO) and polysaccharides
(e.g. chitin) in the wall.
Wang et al. [41] studied the effect of interaction of different carbon-
based materials with two important plant pathogenic fungi: Fusarium
graminearum and Fusarium poae. The strongest antifungal activity
was found in SWCNTs, followed by MWCNTs, GO and rGO, while
C60 and activated carbon showed no significant antifungal activity.
The antifungal mechanism was deduced to target the spores in three
G+ and/or G−
bacteria References
G− Akhavan et al. [35], Perreault et al. [44]
G+, G− Akhavan and Ghaderi [29], Hu et al. [28]
G+, G− Gurunathan et al. [36], Krishnamoorthy et al. [30],
Liu et al. [34], Wu et al. [75], Zhang et al. [90]
−
G Akhavan and Ghaderi [39], de Faria et al. [51], Ruiz et al. [129]
104 A. Lukowiak et al. / Advances in Colloid and Interface Science 236 (2016) 101–112
steps: (1) depositing on the surface of the spores, (2) inhibiting water
uptake, and (3) inducing plasmolysis. Other reports claimed no antifun-
gal activity of GO towards Candida albicans and Candida tropical [42,43].
3. Progress and advances
From the quantitative analysis of scientific data (Fig. 2) it became ap-
parent that interest in the graphene materials used as antibacterial com-
pounds has increased in the last few years. Since 2010, graphene
compounds have been recognized as worthy of interest in the applica-
tion as antimicrobial agents (especially against bacteria). In this section,
we would like to present some recent studies and current research
interests.
3.1. Recent reports on antimicrobial activity
Reports on the biological activity of GO and other graphene family
materials are still limited. Therefore, their properties are continuously
studied to understand the activity and to ensure the development of
safe bio-applications.
Perreault et al. [44] have drawn their attention to GO with different
sheet sizes (average sheet area ranging from 0.01 to 0.65 μm2) and de-
posited on substrate or dispersed in solution. In suspension assays, they
noticed that the antimicrobial effect against E. coli increased with in-
creasing sheet area because GO interacted with bacteria in a cell entrap-
ment mechanism. In contrast, when GO was used as surface coating, a
higher antimicrobial effect of smaller sheets was observed (attributed
to oxidative mechanisms associated with higher defect density of small-
er sheets). This fact emphasizes the importance of choosing the accurate
form of carbon nanomaterial depending on the designed application.
As it was already mentioned, not only physicochemical properties of
graphene-based materials are important when defining antimicrobial
properties but also the facilities of the microorganism cell matter. More-
over, it has been proved recently that there is a direct relationship be-
tween the shape of the nanomaterial and the cell, which has an
impact on antibacterial activity [45]. While diverse graphene quantum
dots (GQDs) with flat sheets lack antibacterial properties, the particles
having nonzero Gaussian curvature (prepared by rupturing C60 cage) ef-
fectively kill S. aureus, including its antibiotic-tolerant persisters, but not
Bacillus subtilis, E. coli, or P. aeruginosa. The observed activity may be cor-
related with the GQD's ability to disrupt the bacterial cell envelope
when the surface-Gaussian-curvatures of a GQD and a target bacterium
match (such as a spherical cell of S. aureus).
Fig. 2. Number of scientific documents interested in graphene itself (graph in blue) and its
interaction with bacteria (graph in red).
According to Scopus, February 2016.
Research performed on large-area monolayer graphene films on Cu
(conductor) and Ge (semiconductor) has surprisingly shown that
these structures can inhibit the growth of E. coli and S. aureus), whereas
the proliferation of both bacteria cannot be significantly restricted by a
graphene film on SiO2 (insulator) [46]. The morphology of cells after di-
rect contact with graphene films on Cu and Ge further confirmed that
membrane damage occurred, while no evident membrane destruction
was induced by graphene on SiO2 (Fig. 3). The authors concluded that
the antibacterial activity of graphene does not stem from ROS mediated
damage (or mechanical damage by sharp edges), but through electron
transfer interaction from the microbial membrane to graphene. Similar
behavior was also found in other carbon allotropes–fullerenes [47].
Numerous studies concern interaction of graphene-based materials
with bacteria strains commonly used for studies, such as E. coli or
S. aureus. However, one can also read about the antimicrobial properties
against other bacteria and pathogens. Good antifungal activity of rGO
sheets against A. niger, A. oryzae, and F. oxysporum was shown by
Sawangphruk et al. [40]. They indicated that for the aforementioned
fungi, MIC50 (the concentration that inhibits 50% of microorganisms)
for pure rGO is equal to 50, 100, and 100 μg/mL respectively. Chen
et al. [38] proposed that GO intertwines with the bacterial and fungal
spores through a wide range of aggregated GO sheets, resulting in a
local perturbation of their cell membrane and inducing a decrease in
the bacterial membrane potential and a leakage of electrolytes in fungal
spores.
Moreover, deoxyribozyme-loaded GO [48] and graphene–tungsten
oxide thin films [49] have been shown to exhibit promising antiviral
properties.
One of the major problems in therapeutics and thorough disinfection
is the biofilm — a form of bacteria life with decreased sensibility to en-
vironmental factors such as antimicrobials. The biofilm, i.e. a group of
microorganisms adhering to surfaces, is surrounded by an extracellular
polymeric matrix of exopolysaccharide that protects microbes against
harmful environmental factors. Therefore, the access of drugs to bacte-
ria cells is strongly limited. One of the important areas where MDR
bacteria may grow and form a biofilm is the endoscopic tools used in
hospitals. Sterilization of endoscopic instruments is problematic due
to their sensitivity to high temperature and chemicals, so the presence
of MDR Salmonella spp., Pseudomonas sp., and E. coli is reported, causing
bacterial infections to transfer between patients [2,50]. Addition of
graphene materials as a carrier (e.g. for silver, antibiotics or biocides)
gives us the possibilities to reduce the number of bacteria and pro-
tects against their adhesion, growth, and biofilm formation [51].
The active layer coated on the protected surface may include, for ex-
ample, graphene oxide and polyvinyl-N-carbazole — both having an-
timicrobial properties and forming an efficient antibacterial and
anti-biofilm composite [52]. Another composition – polyurethane
with polyethylenimine-modified slightly reduced graphene oxide –
exhibited excellent bacterial antiadhesive performance towards
E. coli and it was proposed that the presence of high-density amine
groups in the polyethylenimine chains considerably contributed to
this property [53].
In general, fabrication of different kinds of nanocomposites may
result in obtaining materials with better properties and higher ac-
tivity. This group of materials will be discussed in the following
section.
Finally, to explain the interaction of pristine graphene, its derivatives
and composites with bacteria, recent simulation studies may be helpful,
for example to better understand the contact of bacteria with graphene-
based flat surfaces or nanoparticles (NPs) in suspension [54,55].
3.2. GO functionalization and nanocomposites with graphene family
materials
The antimicrobial properties are related to the form, size, and oxy-
gen functionality of graphene-based sheets, but their activity can be
 A. Lukowiak et al. / Advances in Colloid and Interface Science 236 (2016) 101–112
Fig. 3. SEM studies of E. coli and S. aureus growth onto graphene films deposited on Cu, Ge or SiO2. Images are presented at both low and high magnification. The white arrows at high
magnification correspond to the rectangular areas at low magnification, respectively. Membrane damage is observed for cells seeded onto graphene@Cu and graphene@Ge.
Reprinted from Li et al. [46] with Nature Publishing Group permission.
highly modified through the functionalization with proper groups, mol-
ecules, polymers or nanoparticles. The presence of reactive groups on
the GO (or GrO) surface makes it easy to functionalize with, e.g. (1) an-
tibiotics, (2) chemotherapeutics, (3) immunoglobulins, (4) metals
(nanometals), such as silver and gold, and (5) other organic or inorganic
compounds.
Especially, nanocomposites formed by graphene-based materials
and metal particles are promising effective antibacterial agents with
synergistic action which may be used in a very wide range of both clin-
ical practice and research as well as technical applications typically re-
lated to water purification [56–58].
The most common bacteriostatic nanomaterials are compounds
with silver (ions and/or NPs) which may be incorporated into a carbon
material [59]. Silver shows antibacterial, antifungal, antiprotozoan, and
antiviral activities [60,61] and has been known for its biological activity
for ages (as silver salt AgNO3 and silver sulfadiazine). Its oligodynamic
effect and multilevel mechanism of actions cause a little risk of resis-
tance development. The mechanism of Ag+ activity has been explained,
but the antibacterial activity of silver nanoparticles (Ag NPs) still
causes controversy. Ag action depends on the size, shape, and aggre-
gation of the particles (nanosilver never appears as separated NPs
due to the strong tendency of nanostructures to form clusters). Prob-
ably, the main reasons for the nanosilver antibacterial effect are as
follows: (1) direct contact of Ag NPs with a pathogen cell and interac-
tion with cell wall and membranes, (2) cell penetration and interaction
with inner compounds causing the cytotoxicity effect, (3) genotoxicity,
and (4) ROS generation (with regard to the presence of other molecules,
e.g. TiO2).
105
While Ag NPs in Ag–rGO system have obvious strong antibacterial
activity, rGO was claimed by Xu et al. [62] to be mainly responsible for
adhesion of bacteria cells to this nanomaterial (e.g. as a result of hydro-
gen bonds between lipopolysaccharides and oxygen groups). Thus, rGO
carrier facilitates access of silver to bacteria cells and prevents aggrega-
tion between Ag particles. This was called a capturing–killing process.
Further activity enhancement may be obtained when other components
are introduced in the structure (Fig. 4c). This is the case when chitosan/
Ag/GO nanohybrid is used because chitosan adsorption properties max-
imize the interaction with cells [63].
In the studies of de Faria et al. [51], the GO dispersion showed no an-
tibacterial activity against P. aeruginosa over the concentration range in-
vestigated. On the other hand, the Ag/GO system displayed high biocidal
activity with a minimal inhibitory concentration (MIC) ranging from
2.5 to 5.0 μg/mL. We have proved excellent antibacterial activity of
Ag/rGrO against S. aureus, E. coli, and K. pneumoniae [64] with MIC
values in the range of 4–16 μg/mL. Pure rGrO was inactive at the test-
ed concentration of 512 μg/mL. Similarly, pure GO was inactive
against C. albicans but loaded with silver NPs exhibited antifungal
properties (enhanced also in comparison with Ag NPs) [43]. The au-
thors indicated slightly higher ROS concentration in the medium
after incubation with Ag/GO than with GO. Moreover, the difference
of action depending on the shape of Ag/carbon material was indicat-
ed (see Fig. 4b) showing that carbon nanoscrolls (CNSs) possess en-
hanced and lengthened antifungal activity due to the slow release of
silver ions [42].
The anti-biofilm activity of GO sheets with Ag NPs towards bacteria
cells adhered on stainless steel surfaces has also been reported. The
106 A. Lukowiak et al. / Advances in Colloid and Interface Science 236 (2016) 101–112
Fig. 4. Differences in minimal inhibitory concentration (MIC) values of various graphene-
based materials for selected bacteria and fungi. (a) Antibacterial activity of GrO, GrO with
silver nanoparticles (Ag), and GrO with zirconium phthalocyanine complex (ZrPc) after
5 min near infrared irradiation (Gerasymchuk et al. [82]). (b) Antifungal activity of
GO plates, GO plates with Ag, and carbon nanoscrolls (CNSs) with Ag against
Candida albicans and Candida tropical Li et al. [42]. The inset shows the inhibition
zone of (b-1) GO, (b-2) Ag/GO, and (b-3) Ag/CNSs to C. tropical estimated by the
disk diffusion assay after 24 h incubation; scale bar is 5 mm. (c) Activity of GO,
chitosan/GO, Ag/GO, and chitosan/Ag/GO against methicillin-resistant Staphylococcus
aureus UCLA 8076 (Marta et al. [63]).
(b) The image excerpt reprinted with Elsevier permission.
results showed a 100% inhibition rate of the adhered cells after exposure
to an Ag/GO solution for 1 h [51].
On the basis of assessing the antibacterial effects of metal (Me) NPs
with microscopic techniques, it was found that the cell membrane
damage of bacteria caused by direct contact of the bacteria with Me
(e.g. Cu) is one of the effective mechanisms in bacterial inactivation
[65].
In order to improve water-solubility and long-term antibacterial
activity of rGO, it was modified with poly-l-lysine and copper NPs
[66]. In this case, measurements of K+, Ca2+, Mg2+, and Cu2+ ion con-
centrations in the treated cell extracellular fluid have indicated that
the antibacterial activity of this material can be based on the disrupted
ion concentrations of the bacteria intracellular fluid.
A comprehensive study on GO with different metal or semimetal
NPs was presented by Richtera et al. [67]. The experiment showed the
highest action of Se-doped material against S. aureus and subsequently
lower activity of Ag, AgP, Zn, Cu, and Mn. Similar behavior was observed
for MRSA (both are G+ bacteria) and the effect increased with increas-
ing composite concentrations. On the other hand, the effect of the
same composites on G− bacteria E. coli was observed only at the highest
applied concentration, which further confirmed the difference of re-
sponse between Gram-positive and Gram-negative strains.
rGO decorated with Fe3O4 particles (200–250 nm in diameter) also
has shown high antibacterial properties [68]. Almost 99.6% cells were
viable when treated with Fe3O4 NPs, whereas barely 3% cells survived
in the presence of the composite. The release of ROS oxidative stress
assay was considered as an activity mechanism. In addition, the material
was shown to be a good membrane for the removal of Pb metal ions and
organic dyes from an aqueous solution. Cobalt oxide nanoparticles sup-
ported on rGO sheets are another example of the enhanced action of the
combined components. While GO, rGO, and Co3O4 studied by Alsharaeh
et al. [69] had no antibacterial effect, the Co3O4/rGO showed high activ-
ity against E. coli. Both additives, iron and cobalt oxides, are interesting
also due to their magnetic properties.
3.3. Antimicrobial properties enhanced by photoreactivity
To enhance the antimicrobial activity and therapeutic effects
through photo- and thermotherapy, functionalization of the surface of
carbon materials was successfully provided. Nowadays, photothermal
treatment is a very promising way to ensure aseptic conditions and
heal many microbial infections.
Khan et al. [70] suggested that graphene oxide sheets irradiated
with near infrared (NIR) light (Nd:YAG laser, λ = 1064 nm) have
high antibacterial (S. aureus and P. aeruginosa) and antifungal
(Saccharomyces cerevisiae and Candida utilis) efficacy, higher than
GO without irradiation. The mechanism of the activity was not ex-
plained but local heating and GO reduction were mentioned as possible
reasons for microbe death. Among the obtained results, remarkable
healing of wound infections is very promising, when artificial wounds
created on the dorsal surface of mice were treated with GO and NIR
irradiation [70].
Local heating under NIR irradiation can also be used for selective
killing of pathogenic bacteria when antibody functionalized rGO is pre-
pared [71]. The antibody–rGO complex exhibited little toxicity without
irradiation. However, when irradiated with a NIR laser, it exhibited ex-
cellent photothermal properties and killed captured pathogenic bacte-
ria (S. aureus) specifically.
A graphene-based agent with magnetic properties was also synthe-
sized [72]. Reduced graphene oxide functionalized with glutaraldehyde
and Fe2O3 captured efficiently and killed both G+ S. aureus and G− E. coli
bacteria under NIR laser irradiation. In this material, the advantage of
photothermal properties of rGO was combined with an efficient captur-
ing agent towards bacteria, i.e. glutaraldehyde. Due to their magnetic
characteristics bacteria can be readily trapped in a small volume by an
external magnet and after treatment with NIR, which causes bacteria
death, the composite can be easily removed and reused. In these studies,
graphene demonstrated better photothermal antibacterial efficiency
than carbon nanotubes. Furthermore, a microfluidic chip system under
 A. Lukowiak et al. / Advances in Colloid and Interface Science 236 (2016) 101–112
continuous operation mode demonstrated a biocompatible platform for
online photothermal sterilization.
Various modifications of graphene-based materials were proposed
to create a hybrid photocatalytic material. Titanium dioxide is well
known as a photocatalyst and was used as one of the first compounds
to obtain graphene oxide composite showing photoinduced bacteria in-
activation using solar light irradiation [73].
Nourmohammadi et al. [74] and Wu et al. [75] showed the activity of
GO doped with zinc oxide (ZnO/GO) after visible (Vis) light irradiation.
It was explained that among ROS, •OH radicals were the most responsi-
ble for bacterial inactivation (E. coli K12). Addition of ZnO strongly en-
hanced the photocatalytic effect.
Graphene–tungsten oxide films had an excellent visible light photo-
catalytic performance in the photoinactivation of bacteriophage MS2 vi-
ruses [49]. The interaction of viruses with the surface of the graphene–
WO3 film resulted in a nearly complete destruction of the viral protein
and a sharp increase in the RNA efflux after 3 h visible light irradiation
at room temperature.
Another approach was taken by Liu et al. [76] who functionalized
the GO surface with Ag3PO4 NPs to create a material with strong ab-
sorbance in the Vis region. These particles showed enhanced photo-
catalytic activity as demonstrated by the degradation of a model dye
AO7 through the production of ROS. As a result, strong antibacterial
action against E. coli was observed. The mechanism of action was
suggested to be due to oxidative stress-induced disinfection upon
exposure to visible light.
Cadmium sulfide has also been considered to be a suitable material
for photocatalysis under visible light and sunlight. The disinfection ac-
tivity of a graphene–CdS nanocomposite was evaluated towards E. coli
[77]. The results showed a 1.1-log increase in the inactivation of viable
bacteria after visible light irradiation of CdS and much higher activity re-
sulted in 5.3-log inactivation when exposed to graphene–CdS (10 mg of
each compound was used). In addition, the authors showed that
graphene–CdS toxicity was highly reduced in the presence of humic
acid (HA, a kind of natural organic matter), which may be attributed
to (i) the physical barrier created by HA to prevent contact between
bacteria cells and the material or (ii) the reaction of HA with generated
ROS which reduced their concentration and toxicity.
In our study [60], we have proved excellent antibacterial properties
of TiO2 doped with Ag NPs. On the other hand, TiO2/Ag/rGO composite
presented superior photocatalytic properties as compared to TiO2,
TiO2/Ag and TiO2/rGO (degradation of methylene blue studied by
Vasilaki et al. [78]) and similar enhancement was observed for TiO2/
Fe2O3/Ag/rGO as well (degradation of toxic crystal violet in wastewater
studied by Ghavami et al. [79]). Therefore, it can be expected that TiO2/
Ag/rGO materials also possess much stronger antibacterial properties. In
fact, such enhanced activity was demonstrated by Liu et al. [80] against
E. coli. Eventually, Yang et al. [81] proposed a ternary nanocomposite
(TiO2/Ag3PO4/GO) designed with a synergistic effect. A detailed analysis
of the reaction mechanisms by radical-capture agents suggested that
photo-induced active holes and ROS are responsible for the enhanced
photocatalytic degradation activity owing to effective visible light har-
vesting, improved separation efficiency and fast interfacial charge trans-
fer. Intrinsic antibacterial activity of Ag+ ions may play an important
role in determining the excellent bactericidal performance of this
material.
Recently, we have proposed a novel material where rGrO was doped
with Ag NPs and a complex of zirconium(IV) phthalocyanine (Pc) [82].
The activity of Pcs was previously shown to be useful in diagnostics
and photodynamic therapy (PDT of some viral, bacterial, and fungal in-
fections) where formation of singlet oxygen was observed during expo-
sition to light in the infrared region [83–85]. The antibacterial assay has
shown higher efficacy of the studied Ag/rGrO and Pc/GrO systems
against G− E. coli and P. aeruginosa in comparison to GrO (Fig. 4a). The
activity of all compounds against G+ S. aureus was almost the same.
After 5 min of NIR light irradiation, the antibacterial effect increased
107
significantly for both Ag/rGrO and Pc/GrO. It was clearly seen for the
P. aeruginosa strain, where 128-fold decrease in MIC value was obtained
after irradiation of the Pc/GrO.
4. Hopes and fears
4.1. Toxicity
In biomedical application of all materials the fundamental issues are:
1) high biological (e.g. antimicrobial) activity,
2) lack of cyto- and genotoxicity (biocompatibility),
3) stability.
Safety of the human organism and the environment (animal and
plant population) is crucial. One of the disadvantages of the graphene
family materials is toxicity, which was reported in the literature. Toxic-
ity of carbon nanotubes was widely reported [86], studies on other
graphene-based materials are still in progress. According to the pub-
lished results, cytotoxicity of these materials depends on the concentra-
tion, exposition time, and presence of other factors (e.g. biocompatible
molecules) [87–92].
To kill eukaryotic cells the following ways are possible:
1) contact of the cells with carbon material,
2) induction of ROS,
3) wrapping the cells with nanostructures.
The basic measurements for a cytotoxicity assay are cell viability, cell
adhesion, ROS production, and fluorescence staining.
As in the case of antimicrobial properties, the in vivo studies that have
already been performed on graphene and graphene oxide are sometimes
contradictory and cannot be generalized because of significant variability
of the material under study. According to the data collected by Seabra
et al. [93] graphene-based materials can induce chronic toxicity, dose-
dependent pulmonary toxicity, lung granuloma death, granulomatous le-
sions, inflammatory cell infiltration, pulmonary edema fibrosis, pulmo-
nary inflammatory response, and thrombotoxicity. In contrast, many
graphene materials are considered as non-toxic. Furthermore, special ef-
forts are undertaken to reduce the mortality effect of the graphene mate-
rials through their functionalization, e.g. with amino groups [94], carboxy
groups [95], dextran [96], proteins [87], polyethylene glycol (PEG) [91,
97], phosphorylcholine [88] or polyethyleneimine [92]. Such approach
seems to be particularly interesting in terms of non-cytotoxicity of
graphene-based materials.
Like in the case of bacteria, GO toxicity to eukaryotic cells depends
on the oxidation degree. Zhang et al. [90] proved that lesser oxidation
degree causes higher toxicity of the materials towards the mouse em-
bryo fibroblast (MEF). Studied carbon materials decreased the viability
of the MEF at over 50 μg/mL concentration causing their apoptosis
after 24 h. One may also be concerned about the teratogen properties
of the samples. However, it was not excluded that the mortality effect
of the tested materials was enhanced by the presence of heavy metal
ions, such as Mn2+, in the carbon environment.
Other studies have shown that GO exhibits dose-dependent toxicity
to tissue cells and to animals. Wang et al. [8] have demonstrated that GO
with a dose less than 20 μg/mL did not exhibit toxicity to human fibro-
blast cells, and a dose of more than 50 μg/mL exhibits obvious cytotox-
icity such as decreasing cell adhesion, inducing cell apoptosis, entering
into lysosomes, mitochondrion, endoplasm and cell nucleus. In vivo
tests have indicated no toxicity of GO to mice under a low (0.1 mg)
and middle dose (0.25 mg) and chronic toxicity under a high dose
(0.4 mg) causing mice death and granuloma formation, mainly located
in the lung, liver, spleen, and kidney. Similar conclusion was given by
Chang et al. [98]. Their results suggested that GO does not enter the
A549 cell and has no obvious cytotoxicity but can cause a dose-
dependent oxidative stress in the cell. These effects are both dose- and
size-related.
108 A. Lukowiak et al. / Advances in Colloid and Interface Science 236 (2016) 101–112
The size- and concentration-dependent cyto- and genotoxicity of
graphene oxide sheets and nanoplatelets to the mesenchymal stem
cells was also shown [99]. Small rGO NPs with average lateral dimen-
sions of 11 nm exhibited a strong potential in cell destruction at the
threshold concentration of 1.0 mg/mL, while the cytotoxicity of rGO
sheets with dimensions of 3.8 μm appeared at high concentration of
100 mg/mL after 1 h. Moreover, rGO NPs at low concentrations of
0.1 mg/mL after 1 h could penetrate into the nucleus of the stem cells
and exhibit genotoxicity resulting in DNA fragmentations and chromo-
somal aberrations. No remarkable genotoxicity was observed for big
rGO sheets even at a high concentration of 100 mg/mL after 24 h.
The aspect of the interaction of carbon nanomaterials with human
platelets is important as well [100]. Most of these structures induce ac-
tivation and aggregation of platelets but the activity depends on many
factors. For example, the prothrombotic character of GO was dependent
on surface charge distribution as rGO was significantly less effective in
aggregating platelets [101].
Very recently, a report on the influence of graphene on gut microbi-
ota and their antibiotic resistance genes was published [102]. The re-
sults obtained by the Xie's group are very interesting and worth
emphasizing, because they described the consequences of graphene
nanomaterials usage such as changes in microbiota. A lower graphene
concentration (1 μg/d) had a higher impact on the gut microbiota (in-
creased the biodiversity and changed their community) than a higher
amount (10 and 100 μg/d). It might be due to the aggregation of
graphene particles at the higher content. In consequence, a lower con-
centration probably induced higher oxidative stress and damage of
the cell membrane integrity. Moreover, it was proved that after expo-
sure to graphene, an increase in the types and abundance of antibiotic
resistance genes occurred, which indicates a potential health risk of
graphene.
Waiwijit et al. [89] suggested that graphene added to carbon paste
exhibits a toxicity effect depending on the concentration and exposition
time. They proved that toxicity is proportional to the dose — with lower
graphene level, lower ROS production was observed. Mbeh et al. [87]
studied the mortality of human epithelial cells (A549) after exposure
to graphene oxides nanoribbons doped with albumin at 100 μg/mL con-
centration after 24 h exposition. They established mortality of the
A549 at 58% for this time and dose. A high concentration of graphene
oxide materials doped with proteins (above 100 μg/mL) caused the in-
hibition of human cell growth and induced their apoptosis.
High toxicity of pure graphene oxide may be decreased by the
biofunctionalization of the GO surface with molecules. For example,
according to Liu et al. [88] GO doped with phosphorylcholine did not
indicate cytotoxicity towards murine fibroblast (L929), murine mac-
rophage (RAW 264.7) and human umbilical vein endothelial cells
(HUVEC) after 24 h, and had low cytotoxicity after 48 h.
Unfortunately, functionalization does not exclude toxic behavior of
graphene-based structures. Chowdhury et al. [91] observed differences
in the response of the representative cell lines (Henrietta Lacks cells
(HeLa) derived from cervical cancer tissue, National Institute of Health
3T3 mouse fibroblast cells (NIH-3T3), Sloan Kettering breast cancer
cells (SKBR3), and Michigan cancer foundation-7 breast cancer cells
(MCF7)) to the tested samples of PEGylated oxidized graphene
nanoribbons. Cell viability depended on the GO materials concentra-
tions and type of the cell lines.
As can be concluded from the above results (the in vitro toxicity
effect shown for GO-based material towards cancer lines), one
should consider direct anticancer usage of the graphene materials.
Such usefulness was already reported for modified GO nanoparticles,
e.g. PEGylated nano-rGO for in vivo photothermal cancer (4T1 mu-
rine breast cancer cells) treatment [103] or rGO nanomesh function-
alized with PEG, arginine–glycine–aspartic acid-based peptide, and
cyanine 7 for simultaneous in vivo tumor targeting, fluorescence im-
aging, and photothermal treatment of human glioblastoma U87MG
tumors [104].
Kotchey et al. [105] reported that GO (but not rGO) is susceptible to
the oxidative attack of hydrogen peroxide and horseradish peroxidase.
After 3 months, the graphitic carbon sheets functionalized with polyeth-
ylene glycol (PEG) were cleared and induced no toxicity on mice at a
dose of 20 mg/kg. This observation may enable the design of safer
graphene-based nanosheets that are potentially biodegradable, which
would be of great importance in biological applications such as drug de-
livery [7,106].
Interesting investigations of the biological properties of graphene
oxide-based nanomeshes (GONMs and rGONMs) were performed
when adhesion and proliferation of human neural stem cells on them
as well as stem cell differentiation by NIR laser stimulation were
evaluated [107]. Higher proliferation of the cells on the graphene
nanomeshes than on the GO sheets (or quartz substrate) can be attrib-
uted to better cell attachments on the nanomeshes, due to their better
hydrophilicity (especially the superhydrophilicity of the GONMs) origi-
nating from the excess oxygen-containing functional groups formed on
the edge defects of the particles. Under NIR laser stimulation, the
graphene layers (especially the rGONMs) exhibited significant cell dif-
ferentiations, including more elongations of the cells and higher differ-
entiation of neurons than glia. The higher human neural stem cell
differentiation on the rGONM than the rGO was attributed to the stim-
ulation effects of the low-energy photoexcited electrons injected from
the rGONM semiconductors into the cells, while the high-energy photo-
electrons of the rGO could suppress the cell proliferation and/or even
cause cell damages.
It is worth stressing that the studied materials differ from each other
(e.g. in size, shape, contamination, synthesis method), so, as mentioned
before, the impact of the physicochemical properties of all the materials
on the viability of the advanced organism cells is an important issue. For
instance, the lateral dimension determines the maximum dimension of
the material, which is relevant for cell uptake, renal clearance, blood–
brain barrier transport, and many other biological phenomena that de-
pend on the particle size. Other properties of graphene-based materials
most relevant for their biological effects include surface area, layer num-
ber, surface chemistry and purity, and should be always considered.
Moreover, the morphology, size, and shape of the cell may be significant
in undergoing the apoptosis process.
4.2. Promising applications
All the possible in vivo applications of graphene materials are strictly
dependent on their toxicity. However, there are many other biomedical,
and not only, fields where these systems and their antimicrobial proper-
ties can be very useful despite the possible cytotoxic effect. Sterilization
is one example of such application and others are given below.
Graphene is an ideal material for an electrochemical detector due to
its physicochemical properties — fast electron transfer rate and electro-
catalytic activity. A big advantage of an electrochemical biosensor is its
high sensitivity and fast response in comparison to other detection
methods. Although in such systems carbon structures usually do not in-
teract with microbes directly, they serve as a useful platform to detect a
biological threat.
Chan et al. [108] designed such a biosensor based on reduced
graphene oxide and gold particles (Au/rGO) to diagnose the presence
of botulinum neurotoxin (BoNT) — a virulence factor secreted by
Clostridium botulinum. The presence of BoNT caused the removal of
the peptide immobilized on the rGO surface and changes in the reg-
istered electrochemical current. Rapid identification of this pathogen
based on the enzymatic activity of toxin serotype A, is crucial for botulism
prevention and food safety. A magnetic platform based on multi-
functionalized GO (prepared with N-(4-aminobutyl)-N-ethylisoluminol,
proper antibodies, and Fe 3O 4 NPs), was developed by Sha et al.
[109] to obtain an electrochemiluminescence immunosensor for ul-
trasensitive and rapid detection of marine pathogenic bacterium Vibrio
parahaemolyticus in seawater and seafood.
 A. Lukowiak et al. / Advances in Colloid and Interface Science 236 (2016) 101–112
An electrochemical DNA sensor was fabricated for the detection
of Avian Influenza H5 gene based on graphene/ZnO [110]. The results
obtained from amperometric measurements indicated that the
nanocomposite-enhanced electrochemical DNA biosensor was sig-
nificantly more sensitive and efficient than the conventional agarose
gel electrophoresis. Thus, a proof of the concept was presented that
can be further investigated for the development of an early and
rapid detection tool against the highly pathogenic virus (Avian Influenza
H5N1).
Shi et al. [111] designed a fluorescence resonance energy transfer
(FRET) detector based on GQDs and Au NPs for the detection of
S. aureus by measuring the specific gene sequence. Selectivity, sensitivity
(from 1 nM), and rapid response of the designed biosensor may be used
for the detection of food contamination with S. aureus, causing human
and animal poisoning. Abdelhamid and Wu [112] proposed a fluores-
cence biosensor (based on rGO and flufenamic acid as the active mole-
cule) to determine the concentration of two pathogens – P. aeruginosa
and S. aureus – at excellent linear correlation with the bacteria forming
colony at detection limit down to 3.3 × 103 CFU/mL.
The antibacterial mode proposed in the study of Chen et al. [38] sug-
gests that GO may possess antibacterial activity against multi-resistant
bacterial and fungal phytopathogens and provides useful information
about the application of GO in resisting crop diseases. As an example,
some Xanthomonas perforans bacteria strains can be given. They cause
a disease of tomatoes and are resistant to commonly used bactericides.
Developed DNA-directed Ag NPs grown on GO effectively decrease
X. perforans cell viability in culture and on plants at a very low concen-
tration of 16 ppm and 100 ppm, respectively [113]. Although no phyto-
toxicity was observed in this work, other authors claimed that such
activity was present in their studies [93]. Plants, such as cabbage, toma-
to, red spinach and lettuce, treated with graphene (N500 μg/mL)
showed a significant inhibition of growth, biomass levels, and the num-
ber and size of leaves. The plant cell death induced by graphene treat-
ment might occur either by apoptosis or by necrosis and the toxic
effect depends on the graphene dose, exposure time, and the plant
species.
Carpio et al. [114] designed graphene oxide silanized with
N-(trimethoxysilylpropyl) ethylenediaminetriacetic acid for heavy
metal ions adsorption and reduction, up to ~ 100%, of bacteria cells
(G− Cupriavidus metallidurans CH4 and G+ B. subtilis). Moreover, the
GO–EDTA material did not show the cytotoxicity effect to the human
corneal epithelial cells (more than 99% of the cells stay alive after 24 h).
The application in water purification was mentioned before
[57,68,76,80,81,114]. However, a few layers of GO tend to aggre-
gate under saline conditions thereby reducing its activity. The aggrega-
tion effect can be minimized by having a random arrangement of GO
layers in a three dimensional structure. The study of Jayanthi et al.
[115] emphasized the potential benefits of highly porous, ultralight
GO foams that can be directly used in the removal of aqueous pollutants
(e.g. carcinogenic dyes such as rhodamine B, malachite green, and
acriflavine). The second scope of activity was excellent antibacterial
behavior against E. coli bacteria in aqueous and nutrient growth
media by following the wrapping-perturbation mechanism. A sys-
tem based on Ag/GO plates assembled on a commercial membrane
(cellulose acetate) for biofouling control and water purification was
also prepared [116]. Recent studies provide new insights into the low-
cost, large-scale production of composite materials for application in
degrading harmful organic compounds and common pathogenic bacte-
ria in wastewater.
Graphene materials functionalized with polymers (e.g. chitosan) can
be used for wound dressing, food packaging and coating of various
biomedical devices. The combination of unique properties of two
composite components leads both to a better antibacterial effect and
strongly enhanced mechanical properties [117]. Further studies indicat-
ed that, apart from their bactericidal capacity, chitosan–polyvinylpyr-
rolidone films containing self-organized GO nanolayers possess such
109
characteristics as improved thermal stability, hydrophilicity, water per-
meability, transparency and flexibility [118].
Another described feature can be useful for wound dressing. It has
been found that the peroxidase-like activity of GQDs originates from
their ability to catalyze the decomposition of H2O2 and generating •OH
[119]. The radicals have high antibacterial activity that can be achieved
even at a low concentration of hydrogen peroxide which makes it pos-
sible to avoid the toxicity of H2O2 at high levels in wound disinfection.
The GQD-Band-Aids were successfully prepared showing excellent anti-
bacterial properties (against E. coli and S. aureus) with the assistance of
H2O2 in a low dose.
One more example that can be given here is the antibacterial GO-
modified cotton fabric [120]. Tests have shown that such fabrics possess
strong antibacterial properties and could inactivate 98% of bacteria
(E. coli). Most significantly, they can still kill N 90% of bacteria even
after being washed 100 times. What is more, fabrics caused no irritation
to rabbit skin. Hence, it is believed that these flexible, foldable, and re-
usable GO-based cotton fabrics can be used as a new type of nano-
engineered antibacterial materials for a wide range of applications
(e.g. for bandages).
In another study [121], the synergistic effect of graphene and TiO2
NPs coated on cotton fabric was described. Photo-active GO/TiO2-
modified fabric possessed excellent antimicrobial properties (towards
E. coli, S. aureus, and C. albicans), in contrast to GO-doped cotton that
was inactive. Moreover, the treated cotton samples exhibited low cyto-
toxicity to human fibroblasts.
4.3. Invention disclosure related to antimicrobial graphene-based materials
The usefulness of antibacterial properties of the graphene family ma-
terials has been confirmed by patent claims. Here, the importance of
nanocomposites is clearly seen.
Patented materials include among others: (1) a composite compris-
ing reduced graphene oxide, chitosan, and native lactoferrin [122], (2) a
copolymer of styrene and methylmethacrylate containing reduced
graphene oxide and silver nanoparticles [123], and (3) a hybrid
consisting of graphene oxide covalently conjugated to cationic
quaternized chitosan [124]. These nanomaterials demonstrated a
broad-spectrum antimicrobial activity (against Gram-negative bac-
teria, Gram-positive bacteria, and fungi) and a synergistic effect,
where antimicrobial efficacy of the composites is superior to their
constituent components (GO, rGO, and polymer alone).
The disclosure of Ling et al. [125] relates to the graphene-based
photothermal antibacterial therapy and methods for efficient capture
and killing of bacteria. The authors used the photothermal feature of
rGO for antibacterial activity; in addition, graphene was functionalized
with aldehyde to capture bacteria and with a magnetic material to en-
hance focusing of light irradiation. Moreover, the composition and prep-
aration methods of layers have been claimed, i.e. coatings based on a
polypropylene film covered with a thin layer of oxidized graphene
[126] or nanocomposites including a carbon material (nanotubes or
graphene sheets) and a partial or complete coating of a polymer includ-
ing sufficient π-conjugated moieties to interact with graphene surface
[127].
Ruiz et al. [128] have developed a system and device for filtering
fluids using graphene oxide that can be additionally doped with
nanosilver. GO-based filters may be used for the efficient removal of
microorganisms from organic and aqueous liquids because upon inter-
action, bacteria cells rapidly and irreversibly attach to GO. The afore-
mentioned materials can be also used in implants, medical devices or
food industry.
5. Conclusion
The graphene family materials are certainly very attractive as anti-
microbial agents. In the last 5 years, interest in their usage to reduce
110 A. Lukowiak et al. / Advances in Colloid and Interface Science 236 (2016) 101–112
the number of bacteria cells in different environments has increased sig-
nificantly. Among these materials, graphene oxide is an excellent prod-
uct to be modified or crosslinked with other molecules to further
enhance antimicrobial properties.
In medicine, it can be successfully used for diagnostics, therapeutics
or prevention. Various multifunctional graphene-based nanomaterials
with antibacterial properties can be ideal products used in environmen-
tal pollution protection (e.g. wastewater treatment), food safety, and
other applications, where control of microbe (bacteria, fungi, and even
viruses) growth is essential. Nevertheless, when in vivo application is
considered, one must remember about the possible cytotoxicity effects
that depend on the graphene form, composition, time of exposure,
and the kind of living cells.
Acknowledgments
The authors would like to thank Dr. Yuriy Gerasymchuk for TEM
images and fruitful discussions. This work was partially supported by
Polish Ministry of Science and Higher Education [University of Wroclaw
Grant for Young Researchers no. 0420/1396/1].
References
[1] Rice LB. Federal funding for the study of antimicrobial resistance in nosocomial
pathogens: no ESKAPE. J Infect Dis 2008;197:1079–81.
[2] Ross AS, Baliga C, Verma P, Duchin J, Gluck M. A quarantine process for the resolu-
tion of uodenoscope-associated transmission of multidrug-resistant Escherichia
coli. Gastrointest Endosc 2015;82:477–83.
[3] Taitt CR, Leski TA, Heang V, Ford GW, Prouty MG, Newell SW, Vora GJ. Antimicro-
bial resistance genotypes and phenotypes from multidrug-resistant bacterial
wound infection isolates in Cambodia. J Blobal Antimicrob Res 2015;3:198–204.
[4] Li JL, Tang B, Yuan B, Sun L, Wang XG. A review of optical imaging and therapy
using nanosized graphene and graphene oxide. Biomaterials 2013;34:9519–34.
[5] Notley SM, Crawford RJ, Ivanova EP. Bacterial interaction with graphene particles
and surfaces. In: Aliofkhazraei M, editor. Advances in Graphene Science. InTech;
2013. p. 99–118.
[6] Yang Y, Asiri AM, Tang Z, Du D, Lin Y. Graphene based materials for biomedical ap-
plications. Mater Today 2013;16:365–73.
[7] Goenka S, Sant V, Sant S. Graphene-based nanomaterials for drug delivery and tis-
sue engineering. J Control Release 2014;173:75–88.
[8] Wang K, Ruan J, Song H, Zhang J, Wo Y, Guo S, Cui D. Biocompatibility of graphene
oxide. Nanoscale Res Lett 2011;6:8.
[9] Jastrzebska AM, Kurtycz P, Olszyna AR. Recent advances in graphene family mate-
rials toxicity investigations. J Nanopart Res 2012;14:1320.
[10] Shen H, Zhang L, Liu M, Zhang Z. Biomedical applications of graphene. Theranostics
2012;2:283–94.
[11] Sanchez VC, Jachak A, Hurt RH, Kane AB. Biological interactions of graphene-family
nanomaterials: an interdisciplinary review. Chem Res Toxicol 2012;25:15–34.
[12] Kumar A, Lee CH. Synthesis and biomedical applications of graphene: present and
future trends. In: Aliofkhazraei M, editor. Advances in Graphene Science. InTech;
2013. p. 55–75.
[13] Yang K, Li Y, Tan X, Peng R, Liu Z. Behavior and toxicity of graphene and its func-
tionalized derivatives in biological systems. Small 2013;9:1492–503.
[14] Guo X, Mei N. Assessment of the toxic potential of graphene family nanomaterials. J
Food Drug Anal 2014;22:105–15.
[15] Hummers Jr WS, Offeman RE. Preparation of graphitic oxide. J Am Chem Soc 1958;
80:1339.
[16] Kovtyukhova NI, Ollivier PJ, Martin BR, Mallouk TE, Chizhik SA, Buzaneva EV,
Gorchinskiy AD. Layer-by-layer assembly of ultrathin composite films from
micron-sized graphite oxide sheets and polycations. Chem Mater 1999;11:771–8.
[17] Brodie BC. Sur le poids atomique du graphite. Ann Chim Phys 1860;59:466–72.
[18] Staudenmaier L. Verfahren zur darstellung der graphitsaure. Ber Dtsch Chem Ges
1898;31:1481–7.
[19] Dreyer DR, Park S, Bielawski CW, Ruoff RS. The chemistry of graphene oxide. Chem
Soc Rev 2010;39:228–40.
[20] Botas C, Perez-Mas AM, Alvarez P, Santamaria R, Granda M, Blanco C, Menendez R.
Optimization of the size and yield of graphene oxide sheets in the exfoliation step.
Carbon 2013;63:562–92.
[21] Shen J, Shi M, Yan B, Ma H, Li N, Hu Y, Ye M. Covalent attaching protein to graphene
oxide via diimide-activated amidation. Colloids Surf B 2010;81:434–8.
[22] Mao HY, Lu YH, Lin JD, Zhong S, Wee ATS, Chen W. Manipulating the electronic and
chemical properties of graphene via molecular functionalization. Prog Surf Sci
2013;88:132–59.
[23] Zhou X, Wang X, Wang B, Chen Z, He C, Wu Y. Preparation, characterization and
NH3-sensing properties of reducedgraphene oxide/copper phthalocyanine hybrid
material. Sens Actuators B 2014;193:340–8.
[24] Ghavanloo E, Fazelzadeh SA. Analytical formula to estimate the van der Waals in-
terlayer interaction coefficients for nested spherical fullerenes. Physica B 2015;
478:63–7.
[25] Lu CH, Yang HH, Zhu CL, Chen X, Chen GN. A graphene platform for sensing biomol-
ecules. Angew Chem Int Ed 2009;48:4785–7.
[26] Lee MY, Nam SH, Lee JY, Nahain AA, Lee S, Park CM, Han CJ, Park SY, In I. Formula-
tion of chemically reduced graphene oxide assembly with poly(4-vinyl pyridine)
through noncovalent interaction. J Appl Polym Sci 2013;130:2538–43.
[27] Park S, Mohanty N, Suk JW, Nagaraja A, An J, Piner RD, Cai W, Dreyer DR, Berry V,
Ruoff RS. Biocompatible, robust free-standing paper composed of a TWEEN/
graphene composite. Adv Mater 1736–1740;22:2010.
[28] Hu W, Peng C, Luo W, Lv M, Li X, Li D, Huang Q, Fan C. Graphene-based antibacterial
paper. ACS Nano 2010;4:4317–23.
[29] Akhavan O, Ghaderi E. Toxicity of graphene and graphene oxide nanowalls against
bacteria. ACS Nano 2010;4:5731–6.
[30] Krishnamoorthy K, Veerapandian M, Zhang LH, Yun K, Jae Kim S. Antibacterial effi-
ciency of graphene nanosheets against pathogenic bacteria via lipid peroxidation. J
Phys Chem C 2012;116:17280–7.
[31] Kang S, Pinault M, Pfefferle LD, Elimelech M. Single-walled carbon nanotubes ex-
hibit strong antimicrobial activity. Langmuir 2007;23:8670–3.
[32] Kang S, Herzberg M, Rodrigues DF, Elimelech M. Antibacterial effects of carbon
nanotubes: size does matter! Langmuir 2008;24:6409–13.
[33] Liu S, Wei L, Hao L, Fang N, Wook Chang M, Xu R, Yang Y, Chen Y. Sharper and faster
“nano darts” kill more bacteria: a study of antibacterial activity of individually dis-
persed pristine single-walled carbon nanotube. ACS Nano 2009;3:3891–902.
[34] Liu S, Zeng TH, Hofmann M, Burcombe E, Wei J, Jiang R, Kong J, Chen Y. Antibacte-
rial activity of graphite, graphite oxide, graphene oxide, and reduced graphene
oxide: membrane and oxidative stress. ACS Nano 2011;5:6971–80.
[35] Akhavan O, Ghaderi E, Esfandiar A. Wrapping bacteria by graphene nanosheets for
isolation from environment, reactivation by sonication, and inactivation by near-
infrared irradiation. J Phys Chem B 2011;115:6279–88.
[36] Gurunathan S, Han JW, Dayem AA, Eppakayala V, Kim JH. Oxidative stress-
mediated antibacterial activity of graphene oxide and reduced graphene oxide in
Pseudomonas aeruginosa. Int J Nanomedicine 2012;7:5901–14.
[37] Tu Y, Lv M, Xiu P, Huynh T, Zhang M, Castelli M, Liu Z, Huang Q, Fan C, Fang H, Zhou
R. Destructive extraction of phospholipids from Escherichia coli membranes by
graphene nanosheets. Nat Nanotechnol 2013;8:594–601.
[38] Chen JP, Peng H, Wang X, Shao F, Yuan Z, Han H. Graphene oxide exhibits broad-
spectrum antimicrobial activity against bacterial phytopathogens and fungal co-
nidia by intertwining and membrane perturbation. Nanoscale 2014;6:1879–89.
[39] Akhavan O, Ghaderi E. Escherichia coli bacteria reduce graphene oxide to bactericid-
al graphene in a self-limiting manner. Carbon 2012;50:1853–60.
[40] Sawangphruk M, Srimuk P, Chiochan P, Sangsri T, Siwayaprahm P. Synthesis and anti-
fungal activity of reduced graphene oxide nanosheets. Carbon 2012;50:5156–61.
[41] Wang X, Liua X, Chena J, Han H, Yuan Z. Evaluation and mechanism of antifungal
effects of carbon nanomaterials in controlling plant fungal pathogen. Carbon
2014;68:798–806.
[42] Li C, Wang X, Chen F, Zhang C, Zhi X, Wang K, Cui D. The antifungal activity of
graphene oxide–silver nanocomposites. Biomaterials 2013;34:3882–90.
[43] Cui J, Yang Y, Zheng M, Liu Y, Xiao Y, Lei B, Chen W. Facile fabrication of graphene
oxide loaded with silver nanoparticles as antifungal materials. Mater Res Express
2014;1:045007.
[44] Perreault F, de Faria AF, Nejati S, Elimelech M. Antimicrobial properties of graphene
oxide nanosheets: why size matters. ACS Nano 2015;9:7226–36.
[45] Hui L, Huang J, Chen G, Zhu Y, Yang L. Antibacterial property of graphene quantum
dots (both source material and bacterial shape matter). ACS Appl Mater Interfaces
2016;8:20–5.
[46] Li J, Wang G, Zhu H, Zhang M, Zheng X, Di Z, Liu X, Wang X. Antibacterial activity of
large-area monolayer graphene film manipulated by charge transfer. Sci Rep 2014;
4:4359.
[47] Tang YJ, Ashcroft JM, Chen D, Min G, Kim CH, Murkhejee B, Larabell C, Keasling JD,
Chen FF. Charge-associated effects of fullerene derivatives on microbial structural
integrity and central metabolism. Nano Lett 2007;7:754–60.
[48] Kim S, Ryoo SR, Na HK, Kim YK, Choi BS, Lee Y, Kim DE, Min DH. Deoxyribozyme-
loaded nano-graphene oxide for simultaneous sensing and silencing of the hepati-
tis C virus gene in liver cells. Chem Commun 2013;49:8241–3.
[49] Akhavan O, Choobtashani M, Ghaderi E. Protein degradation and RNA efflux of vi-
ruses photocatalyzed by graphene–tungsten oxide composite under visible light ir-
radiation. J Phys Chem C 2012;116:9653–9.
[50] Kovaleva J, Peters FTM, van der Meic HC, Degener JE. Transmission of infection by
flexible gastrointestinal endoscopy and bronchoscopy. Clin Microbiol Rev 2013;
26:231–54.
[51] de Faria AF, Martinez DST, Meira SMM, de Moraes ACM, Bradelli A, Filho AGS, Alves
OL. Anti-adhesion and antibacterial activity of silver nanoparticles supported on
graphene oxide sheets. Colloids Surf B Biointerfaces 2014;113:115–24.
[52] Mejias Carpio IE, Santos CM, Rodrigues DF. Toxicity of a polymer–graphene oxide
composite against bacterial planktonic cells, biofilms, and mammalian cells. Nano-
scale 2012;4:4746–56.
[53] Tang XZ, Mu C, Zhu W, Yan X, Hu X, Yang J. Flexible polyurethane composites pre-
pared by incorporation of polyethylenimine-modified slightly reduced graphene
oxide. Carbon 2016;98:432–40.
[54] Pham VTH, Truong VK, Quinn MDJ, Notley SM, Guo Y, Baulin VA, Al Kobaisi M,
Crawford RJ, Ivanov EP. Graphene induces formation of pores that kill spherical
and rod-shaped bacteria. ACS Nano 2015;9:8458–67.
[55] Yi X, Gao H. Cell interaction with graphene microsheets: near-orthogonal cutting
versus parallel attachment. Nanoscale 2015;7:5457–67.
[56] Zhang D, Liu X, Wang X. Green synthesis of graphene oxide sheets decorated by sil-
ver nanoprisms and their anti-bacterial properties. J Inorg Biochem 2011;105:
1181–6.
 A. Lukowiak et al. / Advances in Colloid and Interface Science 236 (2016) 101–112
[57] Bao Q, Zhang D, Qi P. Synthesis and characterization of silver nanoparticle and
graphene oxide nanosheet composites as a bactericidal agent for water disinfec-
tion. J Colloid Interface Sci 2011;360:463–70.
[58] Zhu Z, Su M, Ma L, Ma L. Preparation of graphene oxide–silver nanoparticle
nanohybrids with highly antibacterial capability. Talanta 2013;117:449–55.
[59] Das MR, Sarma RK, Saikia R, Kale VS, Shelke MV, Sengupta P. Synthesis of silver
nanoparticles in an aqueous suspension of graphene oxide sheets and its antimi-
crobial activity. Colloids Surf B Biointerfaces 2011;83:16–22.
[60] Kedziora A, Strek W, Kepinski L, Bugla-Ploskonska G, Doroszkiewicz W. Synthesis
and antibacterial activity of novel titanium dioxide doped with silver. J Sol–Gel
Sci Technol 2012;62:79–86.
[61] Kedziora A, Gorzelanczyk K, Bugla-Ploskonska G. Positive and negative aspects of
silver nanoparticles usage. Biol Int 2013;53:67–76.
[62] Xu WP, Zang LC, Li JP, Lu Y, Li HH, Ma YN, Wang WD, Yu SH. Facile synthesis of sil-
ver@graphene oxide nanocomposites and their enhanced antibacterial properties. J
Mater Chem 2011;21:4593–7.
[63] Marta B, Potara M, Iliut M, Jakab E, Radu T, Imre-Lucaci F, Katona G, Popescu O,
Astilean S. Designing chitosan–silver nanoparticles–graphene oxide nanohybrids
with enhanced antibacterial activity against Staphylococcus aureus. Colloids Surf A
Physicochem Eng Asp 2015;487:113–20.
[64] Kedziora A, Geraschymchuk Y, Sroka E, Bugla-Ploskonska G, Doroszkiewicz W,
Rybak Z, Hreniak D, Strek W. Use of the materials based on partially reduced
graphene-oxide with silver nanoparticle as bacteriostatic and bactericidal agent.
Polim Med 2013;43:129–34 [in Polish].
[65] Li H, Chen Q, Zhao J, Urmila K. Enhancing the antimicrobial activity of natural ex-
traction using the synthetic ultrasmall metal nanoparticles. Sci Rep 2015;5:11033.
[66] Ouyang Y, Cai X, Shi QS, Liu L, Wan D, Tan S, Ouyang Y. Poly-L-lysine-modified reduced
graphene oxide stabilizes the copper nanoparticles with higher water-solubility and
long-term additively antibacterial activity. Colloids Surf B 2013;107:107–14.
[67] Richtera L, Chudobova D, Cihalova K, Kremplova M, Milosavljevic V, Kopel P,
Blazkova I, Hynek D, Adam V, Kizek R. The composites of graphene oxide with
metal or semimetal nanoparticles and their effect on pathogenic microorganisms.
Materials 2015;8:2994–3011.
[68] Santhosh C, Kollu P, Doshi S, Sharma M, Bahadur D, Vanchinathan MT, Saravanan P,
Kime BS, Nirmala Grace A. Adsorption, photodegradation and antibacterial study of
graphene–Fe3O4 nanocomposite for multipurpose water purification application.
RSC Adv 2014;4:28300–8.
[69] Alsharaeh E, Mussa Y, Ahmed F, Aldawsari Y, Al-Hindawi M, Sing GK, Alsharaeh E,
Mussa Y, Ahmed F, Aldawsari Y, Al-Hindawi M, Sing GK. Novel route for the prep-
aration of cobalt oxide nanoparticles/reduced graphene oxide nanocomposites and
their antibacterial activities. Ceram Int 2016;42:3407–10.
[70] Khan MS, Abdelhamid HM, Wu HF. Near infrared (NIR) laser mediated surface ac-
tivation of graphene oxide nanoflakes for efficient antibacterial, antifungal and
wound healing treatment. Colloids Surf B 2015;127:281–91.
[71] Wang YW, Fu YY, Wu LJ, Li J, Yang HH, Chen GN. Targeted photothermal ablation of
pathogenic bacterium, Staphylococcus aureus, with nanoscale reduced graphene
oxide. J Mater Chem B 2013;1:2496–501.
[72] Wu MC, Deokar AD, Liao JH, Shih PY, Ling YC. Graphene-based photothermal agent
for rapid and effective killing of bacteria. ACS Nano 2013;7:1281–90.
[73] Akhavan O, Ghaderi E. Photocatalytic reduction of graphene oxide nanosheets on
TiO2 thin film for photoinactivation of bacteria in solar light irradiation. J Phys
Chem C 2009;113:20214–20.
[74] Nourmohammadi A, Rahighi R, Akhavan O, Moshfegh A. Graphene oxide sheets in-
volved in vertically aligned zinc oxide nanowires for visible light photoinactivation
of bacteria. J Alloys Compd 2014;612:380–5.
[75] Wu D, An T, Li G, Wang W, Cai Y, Yip HJ, Zhao H, Wong K. Mechanistic study of the
visible-light-driven photocatalytic inactivation of bacteria by graphene oxide–zinc
oxide composite. Appl Surf Sci 2015;358:137–45.
[76] Liu L, Liu J, Sun DD. Graphene oxide enwrapped Ag3PO4 composite: towards a high-
ly efficient and stable visible-light induced photocatalyst for water purification.
Catal Sci Technol 2012;12:2525–32.
[77] Deng CH, Gong JL, Zeng GM, Jiang Y, Zhang C, Liu HY, Huan SY. Graphene–CdS
nanocomposite inactivation performance toward Escherichia coli in the presence
of humic acid under visible light irradiation. Chem Eng J 2016;284:41–53.
[78] Vasilaki E, Georgaki I, Vernardou D, Vamvakaki M, Katsarakis N. Ag-loaded TiO2/re-
duced graphene oxide nanocomposites for enhanced visible-light photocatalytic
activity. Appl Surf Sci 2015;353:865–72.
[79] Ghavami M, Mohammadi R, Koohi M, Kassaee MZ. Visible light photocatalytic ac-
tivity of reduced graphene oxide synergistically enhanced by successive inclusion
of γ-Fe2O3, TiO2, and Ag nanoparticles. Mater Sci Semicond Process 2014;26:69–78.
[80] Liu L, Bai H, Liu J, Su DD. Multifunctional graphene oxide–TiO2–Ag nanocomposites
for high performance water disinfection and decontamination under solar irradia-
tion. J Hazard Mater 2013;261:214–23.
[81] Yang X, Qin J, Jiang Y, Chen K, Yan X, Zhang D, Li R, Tang H. Fabrication of P25/
Ag3PO4/graphene oxide heterostructures for enhanced solar photocatalytic degra-
dation of organic pollutants and bacteria. Appl Catal B 2015;166–167:231–40.
[82] Gerasymchuk Y, Lukowiak A, Wedzynska A, Kedziora A, Bugla-Ploskonska G, Piątek
D, Bachanek T, Chernii V, Tomachynski L, Strek W. New photosensitive nanometric
graphite oxide composites as antimicrobial material with prolonged action. J Inorg
Biochem 2016;159:142–8.
[83] Buytaert E, Dewaele M, Agostinis P. Molecular effectors of multiple cell death path-
ways initiated by photodynamic therapy. Biochim Biophys Acta 2007;1776:86–107.
[84] Taşkın GC, Durmuş M, Yüksel F, Mantareva V, Kussovski V, Angelov I, Atilla D.
Axially paraben substituted silicon(IV) phthalocyanines towards dental pathogen
Streptococcus mutans: synthesis, photophysical, photochemical and in vitro proper-
ties. J Photochem Photobiol A 2015;306:31–40.
111
[85] Li XS, Guo J, Zhuang JJ, Zheng BY, Ke MR, Huang JD. Highly positive-charged zinc(II)
phthalocyanine as non-aggregated and efficient antifungal photosensitizer. Bioorg
Med Chem Lett 2015;25:2386–9.
[86] Sutariya VB, Pathak Y, editors. Biointeractions of Nanomaterials. CRC Press; 2014.
[87] Mbeh DA, Akhavan O, Javanbakht T, Mahmoudi M, Yahia L. Cytotoxicity of protein
corona–graphene oxide nanoribbons on human epithelial cells. Appl Surf Sci 2014;
320:596–601.
[88] Liu Y, Zhang Y, Zhang T, Jiang Y, Liu X. Synthesis, characterization and cytotoxicity
of phosphorylcholine oligomer grafted graphene oxide. Carbon 2014;71:166–75.
[89] Waiwijit U, Kandhavivorn W, Oonkhanond B, Lomas T, Phokaratkul D, Wisitsoraat
A, Tuantranont A. Cytotoxicity assessment of MDA-MB-231 breast cancer cells
onscreen-printed graphene–carbon paste substrate. Colloids Surf B Biointerfaces
2014;113:190–7.
[90] Zhang W, Yan Y, Li M, Zhao R, Yang X, Ji T, Gu Z, Yin JJ, Gao X, Nie G. Deciphering the
underlying mechanisms of oxidation-state dependent cytotoxicity of graphene
oxide on mammalian cells. Toxicol Lett 2015;237:61–71.
[91] Chowdhury SM, Lalwani G, Zhang K, Yang JY, Neville K, Sitharaman B. Cell specific
cytotoxicity and uptake of graphene nanoribbons. Biomaterials 2013;34:283–93.
[92] Cai X, Lin M, Tan S, Mai W, Zhang Y, Liang Z, Lin Z, Zhang X. The use of
polyethyleneimine-modified reduced graphene oxide as a substrate for silver
nanoparticles to produce a material with lower cytotoxicity and long-term anti-
bacterial activity. Carbon 2012;50:3407–15.
[93] Seabra AB, Paula AJ, de Lima R, Alves OL, Durán N. Nanotoxicity of graphene and
graphene oxide. Chem Res Toxicol 2014;27:159–68.
[94] Singh SK, Singh MK, Kulkarni PP, Sonkar VK, Gracio JJA, Dash D. Amine-modified
graphene: thrombo-protective safer alternative to graphene oxide for biomedical
applications. ACS Nano 2012;6:2731–40.
[95] Sasidharan A, Panchakarla LS, Chandran P, Menon D, Nair S, Rao CNR, Koyakutty M.
Differential nano-bio interactions and toxicity effects of pristine versus functional-
ized graphene. Nanoscale 2011;3:2461–4.
[96] Chowdhury SM, Kanakia S, Toussaint JD, Frame MD, Dewar AM, Shroyer KR, Moore
W, Sitharaman B. In vitro hematological and in vivo vasoactivity assessment of dex-
tran functionalized graphene. Sci Rep 2013;3:2584.
[97] Sahu A, Choi WI, Tae G. A stimuli-sensitive injectable graphene oxide composite
hydrogel. Chem Commun 2012;48:5820–2.
[98] Chang Y, Yang ST, Liu JH, Dong E, Wang Y, Cao A, Liu Y, Wang H. In vitro toxicity
evaluation of graphene oxide on A549 cells. Toxicol Lett 2011;200:201–10.
[99] Akhavan O, Ghaderi E, Akhavan A. Size-dependent genotoxicity of graphene
nanoplatelets in human stem cells. Biomaterials 2012;33:8017–25.
[100] Simak J. The effects of engineered nanomaterials on platelets. In: Dobrovolskaia
AM, McNeil SE, editors. Handbook of Immunological Properties of Engineered
Nanomaterials. World Scientific; 2013.
[101] Singh SK, Singh MK, Nayak MK, Kumari S, Shrivastava SA, Gracio JJA, Dash D.
Thrombus inducing property of atomically thin graphene oxide sheets. ACS Nano
2011;5:4987–96.
[102] Xie Y, Wu B, Zhang XX, Yin J, Mao L, Hu M. Influences of graphene on microbial
community and antibiotic resistance genes in mouse gut as determined by high-
throughput sequencing. Chemosphere 2016;144:1306–12.
[103] Yang K, Wan J, Zhang S, Tian B, Zhang Y, Liu Z. The influence of surface chemistry
and size of nanoscale graphene oxide on photothermal therapy of cancer using
ultra-low laser power. Biomaterials 2012;33:2206–14.
[104] Akhavan O, Ghaderi E. Graphene nanomesh promises extremely efficient in vivo
photothermal therapy. Small 2013;9:3593–601.
[105] Kotchey GP, Allen BL, Vedala H, Yanamala N, Kapralov AA, Tyurina YY, Klein-
Seetharaman J, Kagan VE, Star A. The enzymatic oxidation of graphene oxide.
ACS Nano 2011;5:2098–108.
[106] Liu Z, Robinson JT, Sun X, Dai H. PEGylated nanographene oxide for delivery of
water-insoluble cancer drugs. J Am Chem Soc 2008;130:10876–7.
[107] Akhavan O, Ghaderic E, Shirazian SA. Near infrared laser stimulation of human neu-
ral stem cells into neurons on graphene nanomesh semiconductors. Colloids Surf B
Biointerfaces 2015;126:313–21.
[108] Chan CY, Guo J, Sun C, Tsang MK, Tian F, Hao J, Chen S, Yang M. A reduced graphene
oxide-Au based electrochemical biosensor for ultrasensitive detection of enzymatic
activity of botulinum neurotoxin A. Sens Actuators B 2015;220:131–7.
[109] Sha Y, Zhang X, Li W, Wu W, Wang S, Guo Z, Zhou J, Su X. A label-free multi-
functionalized graphene oxide based electrochemiluminscence immunosensor
for ultrasensitive and rapid detection of Vibrio parahaemolyticus in seawater and
seafood. Talanta 2016;147:220–5.
[110] Low SS, Tan MTT, Loh HS, Khiew PS, Chiu WS. Facile hydrothermal growth
graphene/ZnO nanocomposite for development of enhanced biosensor. Anal
Chim Acta 2016;903:131–41.
[111] Shi J, Chan C, Pang Y, Ye W, Tian F, Lyu J, Zhang Y, Yang M. A fluorescence reso-
nance energy transfer (FRET) biosensor based on graphene quantum dots
(GQDs) and gold nanoparticles (AuNPs) forthe detection of mecAg gene sequence
of Staphylococcus aureus. Biosens Bioelectron 2015;67:595–600.
[112] Abdelhamid HN, Wu HF. A method to detect metal–drug complexes and their
interactions with pathogenic bacteria via graphene nanosheet assist laser
esorption/ionizationmass spectrometry and biosensors. Anal Chim Acta 2012;
751:94–104.
[113] Ocsoy I, Paret ML, Ocsoy MA, Kunwar S, Chen T, You M, Tan W. Nanotechnology
in plant disease management: DNA-directed silver nanoparticles on graphene
oxide as an antibacterial against Xanthomonas perforans. ACS Nano 2013;10:
8972–80.
[114] Carpio IEM, Mangadlao JD, Nguyen HN, Advincula RC, Rodrigues DF. Graphene
oxide functionalized with ethylenediamine triacetic acid for heavy metal adsorp-
tion and anti-microbial applications. Carbon 2014;77:289–301.
112 A. Lukowiak et al. / Advances in Colloid and Interface Science 236 (2016) 101–112
[115] Jayanthi S, Eswar NKR, Singh SA, Chatterjee K, Madras G, Sood AK. Macroporous
three-dimensional graphene oxide foams for dye adsorption and antibacterial ap-
plications. RSC Adv 2016;6:1231–42.
[116] Sun XF, Qin J, Xia PF, Guo BB, Yang CM, Song C, Wang SG. Graphene oxide–silver
nanoparticle membrane for biofouling control and water purification. Chem Eng J
2015;281:53–9.
[117] Yadav SK, Jung YC, Kim JH, Ko YI, Ryu HJ, Yadav MK, Kim YA, Cho JW. Mechanically
robust, electrically conductive biocomposite films using antimicrobial chitosan-
functionalized graphenes. Part Part Syst Charact 2013;30:721–7.
[118] Mahmoudi N, Ostadhossein F, Simchi A. Physicochemical and antibacterial proper-
ties of chitosan–polyvinylpyrrolidone films containing self-organized graphene
oxide nanolayers. J Appl Polym Sci 2016;133:43194.
[119] Sun H, Gao N, Dong K, Ren J, Qu X. Graphene quantum dots-band-aids used for
wound disinfection. ACS Nano 2014;8:6202–10.
[120] Zhao J, Deng B, Lv M, Li J, Zhang Y, Jiang H, Peng C, Li J, Shi J, Huang Q, Fan C.
Graphene oxide-based antibacterial cotton fabrics. Adv Healthcare Mater 2013;2:
1259–66.
[121] Karimi L, Yazdanshenas ME, Khajavi R, Rashidi A, Mirjalili M. Optimizing the
photocatalytic properties and the synergistic effects of graphene and nano titanium
dioxide immobilized on cotton fabric. Appl Surf Sci 2015;332:665–73.
[122] Thalappil, P., Sreenivasan Sreeprasad, T., Maliyekkal Shihabudheen, M., 10.10.2013.
Graphene-based antimicrobial composites. US Patent 20130266628 A1.
[123] Alsharaeh, E.H., Aldosari, M.A., Othman, A.A.R.M., Al-Hindawi, M.F.Q., Alsaud, K.B.B.,
5.03.2015. Novel antimicrobial polymer–graphene–silver nanocomposite. US Pat-
ent 20150065601 A1.
[124] Chan BEM, Li P. Hybrid Nanomaterial of Graphene Oxide Nanomaterial and Cation-
ic Quaternized Chitosan; 18.09.2014[WO 2014142757 A1].
[125] Ling, Y.C., Ramchandra-Deokar, A., Wu, M.C., Liao, C.H., Shih, P.Y., 1.01.2015.
Graphene-based antibacterial therapy and using the same. US Patent
20150004055 A1.
[126] Barkauskas J. Method for Production of Coatings With Antimicrobial Properties;
4.02.2015[EP 2832802 A1].
[127] Advincula, R. 13.01.2015. Polymer nanocomposite precursors with carbon nano-
tubes and/or graphene and related thin films and patterning, US 8932671 B2.
[128] Ruiz, O.N., Fernando, K.A.S., Bunker, C.E., 17.07.2014. Graphene oxide filters and
methods of use. US20140199777 A2.
[129] Ruiz ON, Fernando KAS, Wang B, Brown NA, Luo PG, McNamara ND, Vangsness M,
Sun YP, Bunker CE. Graphene oxide: a nonspecific enhancer of cellular growth. ACS
Nano 2011;5:8100–7.