The dependence of the Graphene-Microbial Interactions
on Graphene Dispersion Fishnet Effects
by
Francis O. Atore
AN HONORS THESIS
for the
UNIVERSITY HONORS COLLEGE
Submitted to the
University Honors College
at Texas Tech University in
partial fulfillment of the
requirement for
the degree designation of
HIGHEST HONORS
MAY 2015
Approved by:
_______________________
Dr. Micah J. Green, Chemical Engineering
_____________
Date
_______________________
Dr. Jaclyn Cañas-Carrell, Environmental Toxicology
_____________
Date
_______________________
Dr. Keira V. Williams, Honors College
_____________
Date
_______________________
Dr. Michael San Francisco, Dean, Honors College
_____________
Date
The author approves the photocopying of this document for educational purposes
Copyright 2015, Francis O. Atore
i
Abstract
Graphene exhibits a unique chemical and physical structure that enables it to have
numerous biomedical applications; however, various researchers have reached different and
conflicting conclusions, and thus the safety and risk of graphene toxicity remains a serious
concern.[1] In order to begin analyzing the cytotoxic nature of graphene, an understanding of
the properties of a graphene sheet in a dispersion is essential.[2] The graphene concentration was
measured, and the results from growing bacteria in graphene dispersions were analyzed
according to the understanding of dispersion, in order to assess whether microbial growth was
dependent on graphene dispersion behavior. The objective of this study is to analyze the
dependence of graphene-microbial interactions on graphene dispersions by supporting the
existence of an entraining effect , or fishnet effect, in graphene dispersions. The concentration is
the main parameter manipulated in studying the forces involved in the fishnet concept, while
entraining is simulated using graphite flakes poured on top of the dispersion. A reduction in
concentration is observed when the centrifugation step is carried out three days after sonication
versus sonication immediately after centrifugation. The entrainment simulation resulted in a 39%
decrease in the concentration of a sample sonicated immediately after sonication and a 5%
increase in a sample centrifuged three days after sonication, thus supporting the existence of a
fishnet effect. The effects of graphene on microorganisms were studied by doing a plate agar
test. The plate agar test resulted in uniform growth across the plate, but no observable toxicity or
"kill zones”. The plate agar test was followed by growing bacteria in a liquid dispersion, in
which an observable film formed at the top, but graphene sheets were observed to be present in
the biofilm. It was also observed that the dispersion had destabilized over time, with sheets
sedimenting to the bottom as the bio-film continued to grow. A crystal violet assay confirmed
that there was a decrease in microbial growth in the presence of graphene, but a trend with
ii
changes in concentration was not observed. These observations, coupled with the data, support
the dependence of graphene-microbial interactions on graphene dispersion effects independent of
concentration. Also, as the sheets fall out of solution, microbial growth persists, indicating the
conversion of few-layer sheets to multi-layer sheets mitigates graphene toxicity. Therefore,
although graphene may reduce the rate or amount of microbial growth, with time, the
destabilization of graphene sheets due to the fishnet effect may mitigate toxicity over time.
iii
Acknowledgments
I would like to thank Dr. Micah J. Green and Dean Michael San Francisco for their
unwavering support throughout my college career and throughout the writing process. I
would also like to thank Dr. Keira W. Williams, a prime example of a motivator and my
mother, Janet Atore, for cheering me on. I would like to thank Dr. Jaclyn Cañas-Carrell
for her insightful comments on toxicology. I also acknowledge my colleagues, Charles
Brandon Sweeney, Fahmida Irin, Dr. Sriya Das, Rozana Bari, Amanda Parra, Dorsa
Parviz, Matthew Hansen, Shane Metzler and Christopher Klaassen who provided
valuable insight in terms of experimental setup and characterization.
iv
TABLE OF CONTENTS
ABSTRACT .........................................................................................................................ii
ACKNOWLEDGEMENTS...................................................................................................iv
TABLE OF CONTENTS ......................................................................................................v
LIST OF FIGURES................................................................................................................vi
CHAPTER 1 ........................................................................................................................1
Introduction ...............................................................................................................1
Graphene yield and dispersing methods review.........................................................3
Graphene microbial interactions and mechanisms.....................................................7
CHAPTER 2.........................................................................................................................19
Fishnet effect experimental setup...............................................................................19
Microbial effect experimental setup...........................................................................20
CHAPTER 3..........................................................................................................................22
The existence of the fishnet effect..............................................................................22
Graphene-microbial toxicity not concentration dependent.........................................23
Discussion ..................................................................................................................26
Conclusion..................................................................................................................29
BIBLIOGRAPHY..................................................................................................................32
v
LIST OF FIGURES
Figure 1: The intersection of microbial interactions and graphene dispersions......................2
Figure 2: The key parameters affecting graphene yield and microbial interactions................3
Figure 3: Interaction of sheets with cell membranes based on sheet size and orientation.......8
Figure 4: Graphene dispersion in water. .................................................................................19
Figure 5: Graphene dispersion centrifugation.........................................................................20
Figure 6: Microbe Incubation in PVP-Graphene-Water dispersion and PVP-Water..............21
Figure 7: Before entrainment, particles on the order of 20 µm and larger were observed using
optical microscopy, while afterwards, observed particles were on the order of 5 µm. ..........23
Figure 8: No kill zones were observed on plate treatments.....................................................24
Figure 9: Crystal violet assay t-test statistical analysis............................................................25
Figure 10: Susceptibility of sheet entrainment by descending multilayer particles due to exposed
surface ......................................................................................................................................27
vi
Chapter 1
Introduction
Graphene nanosheets, or single atomic layers of graphite, are useful for their unique
combination of strength, flexibility, and electrical and thermal properties[3]. Graphene oxide
(GO) is a functionalized form of graphene, with oxygen containing groups on the surface[4]. It is
preferred for its ability to stably disperse, without stabilizers, in aqueous solutions, unlike
graphene. However, observed strength, flexibility, and electrical and thermal properties in GO
are inferior to graphene. Graphene and GO have been found to have numerous biomedical
applications, ranging from scaffolding to biosensors[2].
However, the safety and risk of graphene toxicity remains a serious concern, as findings
from various studies have produced conflicting conclusions[1,5,6,12]. Graphene toxicity is derived
from the nanomaterial's interaction with a biological cell. It has been suggested that the chemical
and physical make-up of the graphene sheet determines its interaction with the biological cell [2,
7]
. Researchers have addressed the existence of conflicting conclusions in graphene toxicity
through the analysis of the presence of artifacts in assays[8,9], while others have analyzed the
difference between functionalized versus pristine graphene[10]. Other studies have analyzed the
effect of multilayer versus few-layer graphene sheets on microbial toxicity[10]; some have gone
as far as stating that graphene and graphene oxide are toxic to microbial cells, but not harmful to
mammalian cells[5,6]. This is contradictory to another study that found GO to be a nonspecific
growth enhancer for bacterial and mammalian cells[1].
The purpose of this thesis is to analyze the divergence in conclusions in the literature
concerning the different findings on graphene toxicity. The underlying parameters that may
explain the variation in conclusions in literature are also studied. The objective of this study is to
1
support the existence of an entraining or fishnet effect in graphene dispersions and to apply the
analysis of the fishnet effect to the results of growing bacteria in a graphene dispersion.
Therefore, the main focus of the thesis is an analysis of the interdependence of microbial growth
and the graphene dispersion dynamics on the conclusion of a graphene-microbial toxicity study.
Figure 1: The intersection of microbial interactions and graphene dispersions
Literature Review
This review of the literature focuses on the main parameters of lateral size and the
number of layers and degree of functionalization by analyzing techniques previously proposed to
improve graphene concentration, sheet size, and number of layers. This review further analyzes
and studies the parameters involved in the dynamics of graphene dispersions. Finally, this review
emphasizes the role that the main parameters (lateral size, number of layers, and degree of
functionalization)
play
in
determining
2
the
cytotoxicity
of
graphene.
Figure 2: The key parameters affecting graphene dispersions and microbial interactions
1.
Graphene yield and dispersing methods review
Graphene dispersion quality and yield are issues fundamental to the application of
graphene in any industry[3]. Pristine graphene is known to be unstable in most solvents, due to its
tendency to aggregate as a result of the van der Waals attraction forces. Van der Waals forces are
weaker than covalent or ionic forces, and include attractive or repulsive interactions between
dipoles. In order to overcome this effect between graphene sheets, some techniques have been
developed to chemically modify graphene, namely the Hummers methods, whereby graphene is
oxidized to graphene oxide[4]. Other methods utilize pristine graphene by dispersing it in the
presence of surfactants, polymers, and π-π interaction stabilizers, among other stabilizers11].
3
Among the factors that affect nanoparticle dispersion yield, sheet size, degree of
functionalization, and number of layers are the most common[2].
Parviz et al. developed a technique in which graphite flakes were added to sodium
dodecyl benzene sulfonate (SDBS), polyvinyl pyrillidone (PVP), and 1-pyrenesulfonic acid
sodium salt (Py-SASS), then tip sonicated for 1 hour, and centrifuged at 5000 rpm for 4 hrs[11].
The resulting dispersion had a yield of 0.22 mg ml-1, 1 mg ml-1 and 1 mg ml-1 respectively. [11]
A flake thickness of single to few-layers and flake length of 2-2.5µm was observed[11, 3].
Compared to existing dipersion techniques, higher stabilizer to graphene concentration ratios
were obtained, while the overall concentration of obtained dispersions were higher.
During pristine graphene dispersion, there are three steps: stabilizer addition, exfoliation,
and centrifugation or separation. As mentioned previously, stabilizer addition is to prevent the
aggregation of graphene sheets due to the attractive van der Waals forces. Exfoliation separates
the raw material, graphite, into its monolayer sheets, graphene. Exfoliation may include
supercritical exfoliation[13] and a variety of sonication techniques ranging from bath[14] to tip
sonication[11]. For the purpose of this thesis, I focus on tip sonication, as it less resource intensive
in terms of time taken and the resulting sheet size is larger. After the sonication step,
centrifugation is used to separate the aggregates from pristine graphene sheets.
During centrifugation, gravitational forces pull multilayer sheets to the center of gravity.
Larger sheets have higher sedimentation rates according to Stokes’ law, after balancing
centrifugal forces against buoyancy and viscous drag.
It has also been shown that when a Brownian particle, such as graphene, has increased
velocity, the hydrodynamic mobility of the particle is reduced due to the inverse relationship
4
with the drag force[15]. The presence of the increased velocity, especially during centrifugation,
increases the drag force, thus increasing the damping force of the fluid on the translational and
rotational axes[15].
Graphene is mobile along three axes, however, two are faster than the other[15]. A
spherical particle usually experiences equal vibration in all axes, thus making it isotropic.
Therefore, if a particle were to experience unequal pull in one direction, this would indicate
anisotropic properties. For a linear nanostructure, anisotropic hydrodynamics would indicate
increased mobility along an axis[15] Graphene flakes have increased fluctuations in longitudinal
and transverse directions, which is attributed to rotational motion, a function of lateral sheet
size(∆) and dynamic viscosity (η)[15]. The property acts as an identifier of the two dimensional
structure of graphene.
Measured "brownian kicks" of the graphene sheets have been shown to only depend on
the lateral size of the graphene sheet and not the thickness, due to the 2D geometry that strongly
affects both hydrodynamics and the radiation force and torque[15,16]. Graphene sheet size and
anisotropy determines the flake stability[15,16]. In order to analyze these properties, dynamic light
scattering, which utilizes the intensity of light scattered by a particle in solution over time, is
used[17]. The particle size and fluid control the hydrodynamic radius[17].
The mean translational diffusion coefficient (D) is obtained by observing the intensity
and scattering angle of the dynamic light scattering signal. A large diffusion coefficient would
indicate a large particle, while a small one would indicate a small particle size. D is then
converted into an apparent hydrodynamic radius Rh through the Stokes-Einstein relation[16]
Rh = kBT/6πηD
5
where kB is the Boltzmann constant, T is the absolute temperature, and η is the viscosity of the
solvent[16].
Afterwards the average lateral nanosheet size can be estimated by using
L = (0.07 ± 0.03) Rh DLS (1.5±0.15)
where L is average lateral nanosheet size[17]. The sheet lateral size determines the sedimentation
rate and also exposes the graphene sheet to entrainment with increasing sheet size.
6
Graphene microbial interactions and mechanisms
1.
Edge functional groups and effects
Graphene has an extreme aspect ratio with a surface area of 2630 m2 for single-layer
graphene[10] and a theoretical thickness of one carbon atom. The sharp edge allows the sheet to
puncture, rupture and damage cell membranes, which could then lead to cell lysis, the release of
vital intracellular content[7], and subsequently cell death.
Pristine graphene is also capable of reactions at its edges due to the presence of
functional groups. Carbonyl, hydroxyl, and epoxy groups have been found along the edges of
holes with surface area measuring under 5 nm2[18]. GO primarily contains carboxylic groups on
its edges and phenol, hydroxyl, and epoxide groups on its basal plane[11]. GO has been found to
have significant catalytic activity for oxidizing biological targets such as glutathione using
oxygen complexes bound on edge and defect sites[19]. Reduced GO contains defects produced
during thermal annealing and removal of the oxygen groups[15]. Defect sites present on GO
mirror those observed on the edges of holes of pristine graphene[18].
It has been found that graphenic carbon surfaces react with dissolved dioxygen to form
surface-bound oxygen containing group intermediates such as lactones, pyrones, ketones,
phenols, lactols and carboxyls[20], which in turn oxidize glutathione (GSH) to glutathione
disulfide (GSSG)[19]. GSH is an important antioxidant present in plant, fungal, animal, and
bacterial cells. It is often used as a biomarker for oxidative stress. Depletion of GSH, or the
reduction in ratio of active reduced GSH to its inactive oxidized form GSSG, is indicative of
reactive oxygen species production[19]. However, this method is vulnerable to GSH adsorption
onto nanoparticle surfaces, which may then be misinterpreted as ROS production or oxidative
stress[19].
7
2.
Lateral dimension and number of layers
The lateral sizes for graphene range from 10 nm to greater than 20 µm, the size of some
cells[21]. The lateral sheet is usually estimated from transmission electron microscopy (TEM) or
DLS. Depending on the lateral dimension of the sheet, the cell may ingest it and experience
membrane integrity disruption or attach to the surface. When a cell attaches to a surface, it may
use the surface as a scaffold, or experience oxidative risk due to nutrient adsorption by the
nanomaterial[21,7,9].
Adsorpti
on
Membrane
Embedded
Puncturin
g
Figure 3: Interaction of sheets with cell membranes based on sheet size and orientation
8
Sheet lateral size and number of layers have been found to alter microbial response in
materials with similar functional groups. For example, GO sheets with an average sheet size of
0.31± 0.20 μm have been found to have higher antimicrobial activity than graphite oxide (GtO)
dispersions with higher average sheet sizes of 6.28 ± 2.50 μm[10].
Smaller diameter sheets have been found to be much more stable in dispersion, which
increases their surface contact per sheet with cells[10]. However, the probability of sheet
internalization by the cell also increases, due to the small diameter (i.e. A546 lung cells
internalizing graphene sheets)[22]. It has also been observed in graphene membrane interaction
simulations that from the time a cell membrane comes into contact with a nanosheet, it may take
a total of 516 nanoseconds for a sheet to be inserted and stabilized in the center of the membrane
bilayer[21].
3.
Surface area and chemistry
Due to the large surface areas of materials such as graphene and GO, phenomena such as
physical adsorption and catalytic surface reactions could affect the biological response to carbon
nanoparticles. High surface area carbon nanotubes, which resemble rolled up graphene sheets,
are known to adsorb molecular probe dyes and in vitro artifacts in toxicology assays and
micronutrients in cell culture[9], prolonging the lag phase in the cell growth cycle and leading to
growth inhibition. Similar effects have also been observed for carbon black[9].
After oxidation of graphene by Hummer's method, the resulting GO material has been
found to be highly inhomogeneous with respect to structure. GO surface consists of holes,
graphitic regions, and disordered regions indicating areas of high oxidation, with holes
composing of 2% of the area, graphitic regions at 16%, and disordered regions at 82%,
9
respectively[25]. As a result, the hydrophobic regions present on GO are minimal, reducing its
adsorptive capabilities.
On the other hand, pristine graphene surfaces are mostly hydrophobic; they poorly
disperse in water and usually require some sort of surfactant or stabilizing agent in aqueous
solutions. The high surface areas of carbon based materials, such as graphene, make surface
reactions potentially important, due to the increased probability of deactivation of antioxidants[24]
and quenching of reactive oxygen species (ROS)[24]. Antioxidants in the body are responsible for
protecting the cells from oxidative damage while ROS are capable of inducing oxidative damage.
Studies suggest that oxygen reduction by carbon begins with chemical adsorption of O2
on carbon active surface sites and formation of superoxide anion radicals, whose formation rate
determines the catalytic activity of carbon in oxidation reactions[19, 14]. It has been found that no
ROS production has been detected when a macrophage cell line was stimulated with purified
single walled carbon nanotubes (SWCNT) to surface reactivity[20,
26]
. However, the aqueous
reaction between O2 and graphenic carbon surfaces is reported to form surface-bound oxygen
intermediates capable of oxidizing antioxidants[19].
Graphene surfaces have been shown to interact with dissolved dioxygen to form surfacebound intermediates, which oxidize GSH to GSSG as the major product and restore the carbon
surface to its original state. It is suggested that the reaction takes place selectively on active sites,
which are graphenic edge or defect sites[19].
Adsorption interactions between amino acids and CNT have been demonstrated to be
weaker on the curved surface in comparison to that of the planar graphene[18]. Experiments
analyzing the trend in interaction energy has found histidine < phenylalanine < tyrosine <
10
tryptophan[18]. The trend in interaction energy is correlated to the molecule's degree of
hydrophobicity (lack of affinity for water). Histidine's interaction energy with graphene is 0.55,
while the interaction energy of tryptophan is the highest at 0.84 eV for graphene[18].
The ability of carbon nanomaterials to deactivate antioxidants through direct surface
reaction involving bound oxygen intermediates has been demonstrated[25]. This route can
contribute to oxidative stress pathways and toxicity for carbon nanotubes, but is not unique to
CNTs. Rather, it is a pathway common to a wide range of graphenic carbon materials that
include carbon black, activated carbons, and graphene oxide[19]. The reaction is dependent on
total surface area and mediated by structural defects[19].
4.
Purity
Pristine graphene might contain residual intercalants (acids or salts) or chemical additives
used to separate the layers in the bulk graphite feedstock[26].
The reagents used in various graphene family nanomaterial (GFN) syntheses include
permanganate, nitrate, sulfate, chromate, peroxide, persulfate, and hydrazine[27]. GO can also
contain lower-molecular-weight oxidative debris that are non-covalently attached to the primary
sheets.
Graphene surface chemistry is similar to carbon nanotubes, in that they are both
composed of sp2 hybridized carbon atoms (arranged in a honeycomb network). Therefore, it is
noteworthy that when using purified nanotubes, no generation of superoxide radicals were found
on the surface from macrophages stimulated with nanotubes.[24] This may be due to one of two
facts. First, in the absence of substantial amounts of metal impurities, nanotubes do not generate
ROS by either mechanism (particle or cell derived); or second, purified multiwall carbon
11
nanotubes (MWCNT) are very effective scavengers of ROS, no matter how such species are
generated[24]. Therefore, if the graphene surface were able to generate superoxide radicals, a
similar effect should be observed for the analogous carbon nanotubes.
5.
Small molecule and ion adsorption
Molecules with conjugated π bonds that impart planarity and allow π-π interactions
preferentially adsorb onto graphene. Due to the large surface areas of materials such as graphene
and graphene oxide, physical adsorption and catalytic surface reactions are among the major
methods of interactions with biological materials. High surface area carbon nanotubes are known
to adsorb onto molecular probe dyes, in vitro artifacts in toxicology assays and micronutrients in
cell culture[27, 9].
In conventional assays such as the DCFH assay, conjugated dye molecules come into
direct contact with graphenic carbon surfaces, and physical adsorption combined with quenching
causes underestimation of surface oxidative activity[19].
Micronutrients containing aromatic and hydrophobic groups such as carbohydrates and
lipids are most susceptible to carbon nanomaterial surface adsorption. However, the challenge in
studying these interactions lies in the fact that dye probes used to study and track these
interactions are prone to adsorption onto the surfaces.
It has been shown that the adsorption forces onto nanomaterials surfaces are so strong,
that they may result in the irreversible adsorption of protein[19]. Folic acid, a vitamin, has an
extended planar conjugated structure capable of π − π interactions with carbon surfaces[9]. The
preferential adsorption of folic acid has been shown to correlate with its very low water
solubility (1.6 mg L−1) compared to pyridoxine HCl (105 mg L−1) and niacinamide (105 mg
12
L−1)[9]. For organic compounds, there is a crude correlation between water solubility and
adsorption sometimes referred to as Lundelius' rule, whereby the greater the solubility, the
stronger the solute-solvent bond and the smaller the extent of adsorption, which reflects the
important role hydrophobic interactions typically play in both phenomena[9].
GO surfaces contain approximately 10% fewer hydrophobic surfaces than pristine
graphene; patches large enough to adsorb smaller micronutrients[9]. Due to the small
hydrophobic area, adsorption of hydrophobic and aromatic micronutrients on GO is much less
than graphene, despite GO's very high total surface area. In addition, due to the fact that the
remaining hydrophobic surface is broken into small patches, the adsorption of large molecules,
as those adsorbed by graphene, may be inhibited. Water clusters that are hydrogen bonded to
those functional groups can also partially restrict solute access to the hydrophobic domains[28].
Studies suggest that oxygen reduction by carbon begins with chemical adsorption of O2
on carbon active surface sites[19]. This implies that anaerobic bacteria grown in their natural
environments are not likely to experience oxidative stress in the presence of carbon
nanomaterials.
A study using bovine serum albumin and tryptophan as well-defined model adsorbates
found that non-covalent adsorption on GO basal planes may account for the deactivation of GO’s
bactericidal activity[23]. Moreover, this deactivation mechanism was shown to be extrapolatable
to GO’s cytotoxicity against mammalian cells. The observations suggest that non-covalent
adsorption on GO's basal planes may be a global deactivation mechanism for GO’s
cytotoxicity[23]. Therefore, the deactivation of GO's bactericidal activity by non-covalent
adsorption explains why GO has been observed as a nonspecific growth enhancer[23,1].
13
Nutrient depletion induced by nanomaterial adsorption is a well recognized reason for
nanotoxicity[22]. Cells in GO-pretreated culture medium have been shown not to experience
nutrient adsorption, thus lacking antimicrobial activity[22]. The observations of GO-pretreated in
culture medium are consistent with Ruiz et. al.’s observations, but contrary to Santos et. al.’s
observations[1,6].
Folic acid depletion by adsorption has been shown to lead to growth inhibition in human
HepG2 cells, which are not capable of producing their own folic acid, leading to a starvation
toxicity mechanism[19]. Reduction in folate levels has been observed at graphene concentration
doses above 1–10 µg/mL (depending on the sheet surface area)[9]. The existence of a starvation
toxicity mechanism in humans indicates that a similar mechanism could account for cytotoxicity
in microbial cultures.
A single adsorption event proceeding to equilibrium can lead to permanent depletion of
nutrients and cell growth inhibition. It is believed that this is best regarded as an in vitro artifact,
since this irreversible depletion mechanism does not accurately model physiological behavior in
vivo where nutrient levels can be replenished[9].
6.
Catalysis of oxidative reactions
Oxidative stress has been commonly reported as an effect of carbon nanomaterials on
biological cells. Nano-surfaces have been shown to generate reactive oxidative species, which
are intermediates along the reduction of oxygen to water and include hydroxide radicals, oxygen
radicals, and peroxide.
It has been found that GO is capable of deactivating antioxidant molecules such as
glutathione by catalyzing their reaction with oxygen[18]. GO has also been implicated in
14
imparting oxidative stress on A549 lung cells at low concentrations, with higher GO levels
exhibiting more reactive oxygen species activity[24]. The number of layers has also been found to
be contributing factor to the oxidative stress effect. The level of oxidative stress has been shown
to increase with fewer layered sheets, with single-layer GO being the most serious[24].
It is speculated that oxidative stress catalyzed by graphene-based materials may follow
one of several paths. The first is ROS mediated oxidative stress, in which oxidative stress is
induced by ROS generated by graphite, graphite oxide, GO, and reduced GO[10]. Alternately,
graphene-based materials may disrupt a specific microbial process by disturbing or oxidizing a
vital cellular structure or component without ROS production[10].
Free radicals such as superoxide anions have been used to assess the oxidation capacity
of graphene-based materials using such methods as the XTT Assay[10]. It has been found that the
amount of free radicals generated in the presence of graphene-based materials is minor,
absolving free radical generation as the main mechanism for microbial inhibition[10].
Carbon-based nanomaterials have been suspected to attack glutathione by oxidation,
therefore minimalizing its antioxidant properties. In graphene, oxidation of glutathione has been
suspected to be time- and concentration-dependent. It has been shown that up to 13.0 ± 4.0% of
GSH can be oxidized by an rGO concentration of 5 μg/mL and up to (99.0 ± 1.1%) of GSH can
be oxidized by an rGO concentration of 80 μg/mL[10]. In GO, an in solution concentration of 5
μg/mL has been shown to oxidize 5.3 ± 2.9% of GSH, while an 80 μg/mL concentration has
been shown to oxidize up to 22.0 ± 0.1% of GSH[10]. It is worth noting that the reduced form of
GO has a higher oxidizing potential, although it has fewer functional groups.
15
In addition, it has been shown that longer exposure time of an antioxidant such as
glutathione has resulted in increased oxidation. After 1 hour of GSH exposure to rGO, 79.9 ± 1.6
of GSH was oxidized, with the percentage increasing to 99.9 ± 1.2% after 4 hours of
exposure[10]. Graphite has been demonstrated to oxidize more GSH than GO, implying that the
level of oxidation of graphene materials also plays a role in their antibacterial activities[10]. Also,
when GO was compared with rGO, GO has demonstrated a much stronger oxidation capacity
toward GSH than rGO[10]. Such a response is attributed to the smaller sheet size of GO, which
has been linked to membrane disruption.
It has been shown that without direct interactions with bacterial cells, the oxidation
capacity of rGO is mitigated[10]. SWCNTs have been shown to act as a conductive bridge over
the cell membrane lipid bilayer, thus mediating electron transfer from bacterial intracellular
components to the extracellular matrix[10]. Therefore, similar surface chemistry materials such as
graphene-based materials may be capable of oxidizing cellular components by acting as a
conductive bridge for intracellular components to the extracellular matrix[10].
Fullerene, an atom thick carbon sheet in a spherical from, is known to attack radicals
since it undergoes radical addition of carbon-centered radicals due to the high electron affinity of
the carbon framework[26]. The mechanism is not similar to the free radical or ROS generation
mechanism proposed in earlier papers. In fact, it acts like an antioxidant, as it eliminates free
radicals.
There are two radical generating mechanisms that have been proposed in the literature:
the generation of HO* radicals in the presence of H2O2, and the generation of CO2 radicals
following cleavage of the C–H bond of formate[26]. The first mechanism mimics the contact of
16
particles with physiological fluids during their phagocytosis, such as in alveolar macrophages
and recruited granulocytes[26]. The second mechanism is a reaction that can be triggered directly
by active sites at the particle surface on contact with the formate ion, or by a short-lived radical
(e.g., hydroxide radicals) which would react with formate[26]. However, neither mechanism
produces a significant amount of free radicals, indicating that this is not a primary method of
carbon nanomaterial interaction with biological cells[26].
It has been found that free radicals are attracted and grafted to the carbon framework of
fullerenes[26]. In addition, MWCNT have been found to be effective scavengers of reactive
oxygen species, both HO* and O2-*, no matter how such species are generated[26]. A similar
mechanism can therefore be applied to the analogous graphene due to similar surface chemistry
in both materials.
It is therefore clear that sheet size, concentration and number of layers are crucial in
analyzing dispersion quality and carbon nanomaterial cytotoxicity. Graphene colloids are
unstable in most aqueous solutions and thus needs a stabilizer to disperse in most aqueous
solvents. In addition, the combination of forces acting on a graphene sheet are integral in
understanding how a sheet may be affected in a dispersion, especially when the entraining effect
is taken into account. If a fast sedimenting particle comes into contact with a slow sedimenting
particle, the fast sedimenting particle may drag the slow sedimenting particle out of solution. The
same parameters that affect \ graphene’s dispersibility in solution may affect the cytotoxicity of
graphene to microbes. The lateral area allows for binding of large molecules and the adsoprtion
of nutrients essential for microbial growth, few layer materials display higher cytotoxicity, while
higher concentrations show stronger cytotoxicity per sheet. The relationship between the
17
entraining effect in graphene dispersions and graphene cytotoxicity is further analyzed and the
convergence in the entraining effect and microbial growth is explored.
18
Chapter 2
1.
Fishnet effect experimental setup
50 mg of graphite powder was added to 50 ml of a stabilizer aqueous solution consisting
of 2 mg/ml pyrenesulfonic acid sodium salt (PY-SASS). The solution was sonicated at 7 W
power for 1 hr. Afterwards, 5 ml of the solution was taken out and left to gravimetrically settle
for 3 days and the absorbance reading and optical microscopy images were taken. Concurrently,
another extract from the sonicated graphite solution was centrifuged at 3600 rpm for 4 hrs to
remove the aggregates. The solution absorbance was then measured and optical microscopy
images were observed for aggregates.
Figure 4: Graphene dispersion in water
19
Graphite powder was added to both solutions, centrifuged, and absorbance readings and
optical microscopy images were taken.
Figure 5: Graphene dispersion centrifugation
2.
Microbial effect experimental setup
Polyvinylpyrrolidone (PVP)-graphene solution was prepared by dissolving 10 mg/ml
PVP in 50 ml of water. Graphite powder was added to the solution and it was sonicated for 1
hour and 7 W power. After this initial sonication, the solution was sonicated at 3600 rpm for four
hours and the absorbance reading was measured. Luria broth was prepared using 10 gm of
tryptone, 5 gm of yeast extract and 10 gm of sodium chloride in 1 liter of de-ionized water. The
solution was then autoclaved and cooled to room temperature.
We then prepared eight luria broth solutions with varying treatments consisting of: pure
luria broth (C1), luria broth innoculated with bacterium (C2), 50 µg/ml graphene, 100 µg/ml
graphene, 200 µg/ml graphene, 300 µg/ml graphene, 400 µg/ml graphene and 500 µg/ml
graphene. Two percent luria broth agar was then prepared by adding 2 wt% agar to luria broth.
Agar plates were then prepared with treatments of 0, 25, 50, 75 and 100 µg/ml.
Crystal violet (CV) assay was prepared using three 24-well tissue culture treated plates
containing 1 ml nutrient broth was inoculated with separate cultures of Escherichia coli, and
20
incubated at 37°C for 24 or 48 hrs. Once the biofilm was observed, the contents of the well plates
were emptied and left to dry in a circulating air hood for approximately 20 min. One ml of a 1%
CV solution in distilled water, filter sterilized using 0.22 µm filters, was added to each well for
45 min to stain the cells. The plates were then emptied and gently washed 5 times with 1200 µl
sterile water to remove any non-attached cells and residual CV. Ethanol (95%, 1200 µl) was
added for 30 min to decolorize the cells. The EtOH/CV solution was pipetted into respective
cuvettes. Absorbance values were recorded spectrophotometrically at OD595, making 1:10
dilutions in ethanol till absorbance was below 1.8. A 1200 µl of 95% ethanol was used as the
blank.
Well C1 was inoculated with Pseudomonas aeruginosa bacteria, while C2 only consisted
of Luria Broth. An equal amount of bacterial culture was added to six wells consisting of
graphene-PVP treatments of 50, 100, 200, 300, 400 and 500 µg/mL. Afterwards, the biofilm
formation in the different treatments was measured using the crystal violet assay. A t-test was
performed to ascertain the statistical trend and significance of obtained data in Table 3.
Figure 6: Microbe incubation in PVP-Graphene-Water dispersion and PVP-Water
21
Chapter 3
Results
1.
The existence of the fishnet effect
To exfoliate graphite into graphene, graphite was sonicated in water and Py-SASS. The
solution was left to settle for 3 days, and a concentration of 0.72 mg/ml was observed (A1).
When A1 was centrifuged after settling for 3 days, a concentration of 0.48 mg/ml was recorded
(A2). The fact that a lower concentration in A2 was observed after centrifugation of A1 indicates
that large aggregates were present in the suspended solution and that there is a potential for large
surface area sheets to be dragged to the bottom by the aggregates during centrifugation. When
graphite powder was added to A2 and centrifuged, a concentration of 0.52 mg/ml (A3) was
obtained. The fact that a slight increase in concentration was observed may be attributed to the
fact that particles in the graphitic powder may have remained suspended in solution. It also
shows that the centrifugation parameters in A2 minimized the existence of aggregates in the
solution. It would, therefore, be an indication of van der Waals attraction between sheets if the
concentration in A2 were to decrease due to centrifugation hours after settling sample A2.
In order to determine if there would be a difference in concentration when there is a delay
between sonication and centrifugation versus having no delay, another experiment was
performed. A dispersion that was centrifuged right after sonication, was measured and a
concentration of 0.69 mg/ml (B1) was obtained. Afterwards, graphite powder was added to
simulate the entraining effect of large particles on graphene sheets and the sample was
centrifuged, resulting in a measured graphene concentration of 0.42 mg/ml (B2). The fact that a
39% decrease in concentration was observed after graphite addition to a freshly centrifuged and
22
sonicated dispersion indicated a presence of the fishnet effect due to larger graphitic particles
dragging few-layer graphene sheets out of solution. Further experiments with Dynamic Light
Scattering (DLS) would need to be performed to confirm the size distribution in the dispersion.
Large surface area sheets are highly susceptible to the fishnet effect due to their exposure to
descending multilayer aggregates.
Gravimetric +Graphite
Normal
+Graphite
Figure 7: Before entrainment, particles on the order of 20 µm and larger were observed using
optical microscopy, while afterwards, observed particles were on the order of 5 µm.
2.
Graphene-microbial toxicity not concentration dependent
A 1 mg/ml graphene-PVP dispersion coating was applied onto a bacteria inoculated
solidified agar surface after bacteria was spread onto the surface. The dispersion was applied in
increments of 25 µg/ml around plate sections. The plates were then incubated at 30°C for 24 hrs.
No kill zones were observed on the plates; in fact, the biofilm was uniformly distributed on both
areas that were and that were not coated with the graphene dispersion.
23
Figure 8: No kill zones were observed on plate treatments
Multilayer graphene has properties different from single-layer graphene due to the
reduced surface area. The effect of the nutrient adsorption mechanism cited in earlier studies is
also mitigated in our agar plate study due to the fact that there is a much higher concentration of
nutrients relative to graphene sheets. Therefore, the nutrient environment is never completely
depleted.
A crystal violet assay was also performed to quantify the bacterial concentration in six
well treatments of 0, 50, 100, 200, 300, 400 and 500 µg/ml of graphene. Compared to the 0
µg/ml of graphene treatment (C1) innoculated by bacteria, the other six treatments had anywhere
from 30 to 25% less bacterial growth relative to C1. The results of the experiment suggest that
graphene is capable of decreasing biofilm formation. However, we were unable to observe any
trends with respect to concentration.
24
Figure 9: Crystal violet assay t-test statistical analysis
From observing the bacteria in liquid nutrients, I was able to notice that the biofilm
formed in the presence of graphene was more deformed and fragmented than that formed in its
absence. The sheets were embedded in the biofilm, a mechanism that has been suggested in
previous studies[7, 21]. It is still not clear whether the sheets in our studies were strictly adsorbed
onto the surface or if some of the sheets may have been intercalated within the membrane of the
bacterial cells.
It is therefore clear that the mode of graphene-microbial interaction is also dependent on
the phase of the media (liquid or solid). In solid media, no kill zones were observed and bacteria
grew uniformly across the graphene coated plate surface. However, when in the liquid phase,
biofilm formation was reduced by 25-30%, due to the uniform dispersion of graphene sheets in
solution.
When the fishnet observations are applied to our observations of the biofilm formation in
the liquid media, we are able to explain why some sheets remain embedded in the biofilm while
some crash out of solution. It is evident that particles in a dispersion lose stability over time. As a
25
result, those present at the top that are trapped in the biofilm would have to be trapped in the
initial stages of the dispersion. In addition, an analysis of size distribution in the biofilm would
shed light on whether sheets present in the biofilm are mostly large or small lateral size.
Graphene-PVP dispersions are known to have a broad pH range, as low as 2[11], therefore pH
alterations due to microbial growth would not explain the crashing of graphene from solution.
Future experiments will need to determine relationships between microbial growth over time and
graphene aggregation and size distribution in solution. The formation of aggregates over time
resonates with observations similar to our fishnet experiment in which sheets were dragged to the
bottom by graphitic particles.
Discussion
1.
Fishnet effect
The fishnet theory states that once a graphite-solvent-stabilizer solution is sonicated,
there are varying lateral size and sheet thicknesses floating in solution. Upon setting the
dispersion in an undisturbed environment, multilayer sheets settle faster than few-layer sheets
and in the process, they end up dragging the few-layer sheets out of solution to the bottom
sediment. Among the important factors in this theory are the lateral size of the sheets and the
amount of layers in a sheet. The lateral size increases the surface area of the sheet, exposing it to
fast settling particles, while the number of sheet layers affects its settling rate.
26
Figure 10: Susceptibility of sheet entrainment by descending multilayer particles
We therefore propose that the descending sheets trap the sheets below them while they
descend resulting in the reduction of graphene yield. It has been shown that as graphene remains
suspended in solution over time, the graphene stabilizer interactions are reduced. As the sheets
interaction increased, the sheet stability is reduced, leading to agglomeration and formation of
multilayer sheets.
2.
Graphene-microbial toxicity
The results show that biofilm formation is disrupted in liquid media, due to observed
fragmentation of the film. A physical interaction mechanism may account for the observed
fragmented morphology, as the film may wrap around individual sheets as shown in Liu et al.[10],
thus depriving the cell nutrients initially. However, we noticed that sheets crashed out of solution
over time, and as a result, the concentration of graphene sheets in the dispersion was reduced. It
would therefore make sense for the bacteria to gain access to nutrients and grow much faster as
the graphene concentration around them is reduced, if the cells were not starved out by the initial
adsorption. A more in-depth analysis should be performed on a time lapse of bacteria growing in
a graphene dispersion, in which the sheet size and distribution at different depths is analyzed
27
before and after microbial growth. The growth rate of the film over time, relative to the
concentration in dispersion would explain the adverse effects the fishnet effect would have in
terms of reducing bacterial growth inhibition.
We were unable to see any quantifiable trends with respect to graphene concentration
treatments. However, a significant difference in microbial growth was observed between the
presence and a lack of graphene sheets in solution.
3.
Microbial activity dependence on dispersion quality
A reduction in sheet concentration in a graphene dispersion supported the existence of a
fishnet effect. We also observed that sheets aggregates were present. Aggregation of single layer
sheets results in the creation of more multilayer graphene that could acts as "boulders," according
to the proposed fishnet theory.
The stability of graphene dispersions affects microorganisms in terms of number of layers
and sheet size, both of which affect the adsorption properties. Also, certain microorganisms are
known to release by-products capable of altering the pH of solutions. The altered solution pH is
capable of destabilizing graphene sheets, which in turn could change the nanoparticle microbe
interaction.
Carbon nanomaterial dispersions are sensitive to salts[
9, 19]
, which are known to be
present in some cell growth culture media. The presence of salt alters the stability of the solution
, which may lead to its transition from few-layer graphene to multilayer graphene and possibly
result in graphite. All three materials have distinctly different properties.
28
Graphite has been shown to have a higher oxidation capacity than GO, while GO has a
higher oxidation capacity than rGO[10]; indicating that functionalization and sheet size influence
toxicity results. In addition, the oxidation potential of these materials has been shown to increase
with time[10]. The increase in oxidation potential over time is applicable to the analogous
graphene. As a result, we are more likely to observe bioaccumulation and fewer oxidative
reactions associated with graphene surface reactions.
When graphite powder has been sonicated and dispersed in water in the absence of a
solvent and left to agglomerate over time in the presence of mammalian cells, no loss of cell
viability has been observed[10]. As a result, if a graphene dispersion was left suspended over time
and we observed agglomeration, we would expect to see reduced cytotoxicity. It is possible that
in vivo experiments would indicate bioaccumulation, however, it would be difficult to study
bioaccumulation effects in vitro.
Conclusions
The dependence of graphene-microbial interaction on graphene dispersions was studied
by supporting the existence of an entraining effect, the fishnet effect, in graphene dispersions. A
reduction in concentration was observed when the centrifugation step was carried out three days
after sonication versus sonication immediately after centrifugation. It was also observed that the
entrainment simulation resulted in a 39% decrease in the concentration of B1 and a 5% increase
in A2, thus supporting the existence of a fishnet effect. The plate agar test resulted in no
observable cytotoxicity or "kill zones," but rather in uniform growth across the plate. Bacteria
grown in a liquid dispersion showed that an observable film was formed at the top with graphene
sheets present and that the dispersion had destabilized over time, with sheets sedimenting to the
29
bottom as the bio-film continued to grow. A crystal violet assay confirmed that there was a
decrease in microbial growth in the presence of graphene, but I was unable to observe a trend
with changes in concentration. Observations, coupled with the data, support the dependence of
graphene microbial interactions and graphene dispersion with the exception of concentration. It
was observed that as the graphene sheets fall out of solution, microbial growth persisted,
indicating that the conversion of few-layer sheets to multilayer sheets mitigated graphene
toxicity. Therefore, although graphene exhibits cytotoxicity, the effects may be mitigated by the
colloidal instability of graphene in dispersion.
30
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