Graphene and carbon nanotube composite enabling

Materials Science and Engineering C 41 (2014) 65–69
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Materials Science and Engineering C
journal homepage: www.elsevier.com/locate/msec
Graphene and carbon nanotube composite enabling a new prospective
treatment for trichomoniasis disease
H. Zanin a,⁎, A. Margraf-Ferreira b, N.S. da Silva b, F.R. Marciano c, E.J. Corat a, A.O. Lobo c
a
b
c
National Institute for Space Research, Av. dos Astronautas 1758, Sao Jose dos Campos CEP: 12227-010, SP, Brazil
Laboratory of Cell Biology and Tissue, Institute of Research and Development, University of Vale do Paraiba, Av. Shishima Hifumi, 2911, CEP: 12244-000 Sao Jose dos Campos, SP, Brazil
Laboratory of Biomedical Nanotechnology, Institute of Research and Development, University of Vale do Paraiba, Av. Shishima Hifumi, 2911, CEP: 12244-000 Sao Jose dos Campos, SP, Brazil
a r t i c l e
i n f o
Article history:
Received 20 September 2013
Received in revised form 23 January 2014
Accepted 7 April 2014
Available online 22 April 2014
Keywords:
Graphene oxide
Carbon nanotube oxide
Composite
Tritrichomonas foetus
Drug carrier
a b s t r a c t
We report the synthesis and application of novel graphene oxide and carbon nanotube oxide (GCN-O) composite.
First, pristine multi-walled carbon nanotube was prepared by chemical vapour deposition furnace and then exfoliated and oxidised simultaneously by oxygen plasma etching. The superﬁcial and volumetric compositions of
GCN-O were measured by XPS spectroscopy and EDX spectroscopy, respectively. Both XPS and EDX analyses evidence that the GCN-O is composed of up to 20% of oxygen atoms. As a result, GCN-O forms a stable colloidal
aqueous solution and shows to have strong interaction with the cell membrane of Tritrichomonas foetus protozoa,
making easy its application as a drug carrier. Trichomoniasis infection of cattle is a devastating disease for cattle
producers, causing some damages to females and fetus, and the abortion is the most serious result of this disease.
There is no effective treatment for trichomoniasis infection yet. Therefore, new treatment, especially one with no
collateral effects in animals, is required. With this goal in mind, our results suggest that water dispersible
composite is a novel nanomaterial, promising for Trichomoniasis infection treatment and as therapeutic delivery
agent as well.
© 2014 Elsevier B.V. All rights reserved.
1. Introduction
Recently graphene is at the centre of a signiﬁcant research effort
with the promise of owning properties as good as or even better than
the nanotubes [1]. In the ﬁrst studies, researchers dealt with the preparation and application of graphene, graphene oxide and reduced
graphene oxide [2–6]. The most used method for graphene production
is known as Hummer method [7], which consists of chemical intercalation of metal and abrupt thermal expansion of pristine graphite, which
promotes their exfoliation. Another attractive possibility for graphene
production consists of opening carbon nanotubes, which could be understand as graphene nanosheets rolled up [5]. The multi-walled carbon
nanotubes (MWCNTs) can be opened longitudinally by intercalation of
metals and exfoliation with acid treatment and abrupt heating. The
resulting material consists of: (i) multilayered ﬂat graphitic structures
(nanoribbons), (ii) partially open MWCNTs, and (iii) graphene ﬂakes.
However, both methods describe above need strong chemical oxidant
manipulation, which need special handle care and correct disposal.
Into this context, the oxygen plasma etching is a simple, fast, selective
and low temperature carbon nanotube exfoliation. The ﬁnal product of
carbon nanotube exfoliation is graphene oxide and carbon nanotube
oxide composite. Both graphene and carbon nanotubes are special
⁎ Corresponding author. Tel./fax: +55 1232086558.
E-mail address: [email protected] (H. Zanin).
http://dx.doi.org/10.1016/j.msec.2014.04.020
0928-4931/© 2014 Elsevier B.V. All rights reserved.
materials because of their superb physico-chemical properties such as
biocompatibility, absence of mutagenic and recombinagenic activity,
large speciﬁc surface area and potentially low manufacturing cost
[8–11]. Carbon nanotubes have been extensively studied in recent
years and many technologies were developed using this material
[12–14]. There is a great expectation that graphene has similar
physico-chemical properties compared to carbon nanotubes [15–17],
and should be as good as or better than carbon nanotubes in some applications. However, all those applications should be carefully tested for
graphene as well [18–20]. Considering that, we have shown in
recent publication the strong Tritrichomonas foetus adhesion on to
superhydrophylic vertically aligned carbon nanotube (VACNT) surface
[21]. That study enabled to apply superhydrophylic VACNT to understand protozoa spreading mechanism and the speciﬁc recognition of adhesion proteins. We especially pay attention on this protozoan, because
it causes the trichomoniasis, which is a sexually transmitted disease, affecting male and female cattle. Trichomoniasis infection of cattle is a
devastating disease for cattle producers [22]. The trichomoniasis can
cause some damages to females and fetus, and the abortion is the
most serious consequence [23]. When diagnosed in a herd, it causes
economic loss and emotional pain [24]. Although there is no treatment
for trichomoniasis infection yet [24], there are a couple of reports showing decrease of the protozoa content with estradiol-treatment [25,26].
Studies using ultra-structural cytochemistry and determination of the
cellular electrophoretic mobility showed that T. foetus possesses a net
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H. Zanin et al. / Materials Science and Engineering C 41 (2014) 65–69
negative surface charge [27], which plays a fundamental role on their
ability to adhere to inert and biological substrata.
In this work, we study a novel method to prepare graphene by
oxygen plasma etching of MWCNTs. We propose a novel composite of
graphene oxide and carbon nanotube oxide (GCN-O), exfoliating nanotubes. Further, we evaluate this new material using scanning and transmission electron microscopy; Raman spectroscopy, energy-dispersive
X-ray spectroscopy and X-ray photoemission spectroscopy; zeta potential; surface tension tests and semi-quantitative adhesion to parasitic
protozoan T. foetus.
were conducted at room temperature. The GCN-O zeta potential was
measured by dynamic light scattering (DLS) (Zetasizer, Malvern —
UK). All samples were diluted at 150 μg ml− 1 in KCl 1 mM solution
and were deagglomerate with two different ways: i) conventional sonication during 30 min and ii) using a ultra power sound irradiation (UI)
at 200 W during 30 min. Then samples were placed to a polystyrene
zeta cuvette. Measures were performed 2 h after re-suspension. The
EasyDyne tensiometer (Kruss, K20 model) was used to measure the surface tension of the GCN-O, T. foetus and its conjugate using the Du Noüy
Ring method [31] for an average of 10 data sets.
2. Experimental
3. Results and discussion
2.1. Synthesis of MWCNT and GCN-O
Fig. 1(a–g) shows the typical morphology of GCN-O. Fig. 1(a) is
the top view of GCN-O, which is magniﬁed at higher resolutions
(Fig. 1(b–g)) to better visualisation of the effect of the oxygen plasma
etching at MWCNT. From all those SEM micrographs, we could see
that oxygen plasma exfoliated preferentially the carbon nanotube tips,
opening its walls and disclosing its fundamental structure: graphene
[32-34]. It is well known that carbon nanotubes are composed of
graphene nanosheets rolled into cylinders. In addition, graphene nanosheet rolling up into a tube is a standard picture to explain the carbon
nanotube formation. In that tubular structure, the nanotube tips are
the most defective and are more suitable to interact direct with plasma,
causing the pitch of corrosion and then starting exfoliation from there.
Fig. 1(h) shows the TEM micrograph of GCN-O, which the exfoliation
started from the tip and continued. We noted that depending on plasma
conditions we could etch the whole nanotubes or simply attach oxygen
groups to them without exfoliation. At higher plasma pressures (higher
than 180 mTorr) or for longer processing times, the erosion was completed. With diluted plasma, exfoliation does not show up but wettability increased signiﬁcantly because of oxygen groups' attachment [40].
The GCN-O composite was achieved at quite speciﬁc conditions. Further, all those micrographs point out GCN-O as porous structure,
which the real surface area and speciﬁc density were measured by
BET and helium pycnometry to be ~ 38.5 m2/g and ~ 2.3 g/cm3,
respectively.
Fig. 2(a & b) shows ﬁrst- and second-order Raman scattering spectra
of MWCNT and GCN-O. The deconvolutions were performed using
Lorentzian shapes for the D, G and G′ bands, and Gaussian shape
for bands around 1250, 1480 and 1611 cm− 1 (D′ shoulder) [33,35].
The D band is usually assigned to the disorder and defect of the carbon
crystallites [36]. The G band is assigned to one of the two E2g modes corresponding to stretching vibrations in the basal plane (sp2 domains) of
single crystal graphene [37]. The high intensity G′ band reveals that
these materials present high crystallinity [38]. In the GCN-O ﬁrst order
Raman, for set aside deconvolution ﬁtting, two Gaussian peaks centred
at around 1250 and 1480 cm−1 were added necessarily. Probably the
shoulder has its origin in double resonance, because its Raman shift
(~1200 cm−1) is a point on graphene phonon dispersion curves [39].
The origin of the 1480 cm−1 band is probably correlated with the
polar group grafting on to GCN-O surfaces [40].
We identiﬁed and semi-quantiﬁed chemical elements and chemical
bond in as-grown MWCNT and GCN-O samples using two different
techniques. Fig. 3(a–d) presents the results that we have taken from
as-grown MWCNT and GCN-O by XPS and EDX measurements for an average of 5 data sets. Fig. 3(a & b) shows the (a) C1s and (b) O1s ﬁtted
photoemission spectra recorded and deconvoluted for GCN-O. The spectra were deconvoluted assuming a Lorentzian–Gaussian sum of functions (20% Lorentzian maximum contribution) by Fityk software [41].
Fig. 3(a) presents the C1s spectrumdecomposed into ﬁve Gaussian components, referring to the bonds: sp2 carbon (~ 284.5 eV), C\O
(~ 285.8 eV), C_O and \COO\ (288.7 eV), and the last one at
291.2 eV assigned to the shake-up peak (π–π* transitions) [42,43].
Fig. 3(b) shows deconvolution of the O1s spectrum using three bands.
The ﬁrst is in the range from 531.0 to 532.4 eV, attributed to hydroxyl
The MWCNTs were prepared using a mixture of camphor (85 wt.%)
and ferrocene in a thermal chemical vapour deposition (CVD) furnace,
as reported elsewhere [28]. The mixture was vapourised at 220 °C in a
pre-chamber and then, the vapour was carried by an argon gas ﬂow
at atmospheric pressure, to the chamber of the CVD furnace set at
850 °C. The time elapsed during the process used to produce the
MWCNTs was only a few minutes. The sample was etched by chloridric
acid at 100 °C for 5 h for residual catalytic iron particle removal. And
then, it was extensively water washed and ﬁnally dried. The incorporation of oxygen-containing groups was carried out in a pulsed-direct current plasma reactor with an oxygen ﬂow rate of 1 sccm (standard cubic
centimetres per minute), at a pressure of 185 mTorr, −700 V and with
pulse frequency of 20 kHz [29]. We have developed an apparatus that
shakes and deagglomerate the carbon nanotube powder during oxygen
plasma etching. This apparatus is a hollow cathode with a helix on the
base for powder shaking and grinding. The plasma forms inside this
hollow cathode, allowing the plasma to access three-dimensionally
the nanotube powder in motion for up to 1 h.
2.2. GCN-O and parasitic protozoa T. foetus in TYM medium
The K strains of T. foetus, isolated from the urogenital tract of a bull
were maintained in TYM Diamond's medium (medium of trypticase,
yeast extract, maltose, and serum used to detect the presence of
Trichomonas vaginalis), supplemented with Fetal bovine serum (10%,
Gibco/BRL) at 37 °C [30]. A colloidal solution of 104 T. foetus K was
cultivated during 24 h (logarithmic growth phase) and used in each
experiment with GCN-O. A stock solution (1 mg/ml) of GCN-O composite was prepared in phosphate-buffered saline solution (PBS). A suspension of T. foetus (104 protozoa/ml) was aliquoted into individual wells of
a microdilution plate (24 wells) and 10 μg/ml or 100 μg/ml of GCN-O
composite was added and the mixture was incubated at 37 °C for 1 h.
The adhesion was evaluated by an inverted optical microscope (DMIL
— Leica) and the SEM microscopy. Control groups (protozoa without
GCN-O2 and GCN-O2 only) were performed as well.
2.3. Characterisation of GCN-O composite
The composite was characterised by several techniques such as: high
resolution scanning electron microscopy (SEM), transmission electron
microscopy (TEM), Raman spectroscopy, X-ray photoemission spectroscopy (XPS), and energy-dispersive X-ray (EDX) spectroscopy, and
zeta potential analysed by dynamic light scattering (DLS). The agglomeration status was analysed by inverted optical microscope (DMIL —
Leica). Morphological analyses were performed with SEM-FEG (JEOL
6330) operated at 20 kV and coupled with an EDX for chemical analysis,
operating with a Si (Li) detector with an energy resolution of 126 eV.
Raman spectra were recorded using a Renishaw microprobe, employing
an argon laser for excitation (λ =514.5 nm) with a laser power of approximately 6 mW. The XPS spectra were taken using VSW H100 spectrometer with residual pressure less than 10−10 Torr. All measurements
H. Zanin et al. / Materials Science and Engineering C 41 (2014) 65–69
67
Fig. 1. (a–g) SEM and (h) TEM micrographs of the GCN-O sample.
bound to carbon, the second in the range from 534.6 to 536.1 eV, attributed to oxygen singly bound to carbon and third at 534 eV, attributed to
carboxyl [44]. With our approaches, the oxygen content was estimated
up to 21%, after the oxygen plasma treatment, which was calculated as
performed by Payne et al. [45] method. We compared these results to
those we extracted from EDX spectra. Fig. 3(c & d) shows the spectra
and tabulated the extracted data (inset box) from (c) as-grown
MWCNT and (d) GCN-O samples. One can note that after acid and plasma etching the iron content decreased from 9 to 0.3% of the total atom
content and the oxygen content increased from 1.6 to 18.2% of the
total atom content [46]. Both XPS and EDX analyses evidence that the
functionalised GCN-O is composed of ~ 20% of oxygen. Considering
that the XPS is more appropriated to measure superﬁcial composition
and EDX is more appropriated to evaluated volumetric composition,
the coherence of this results suggests that the GCN-O was three dimensionally functionalised with oxygen content groups. In summary, we
conﬁrmed here our previous works [40,46-49] that the oxygen plasma
can attach oxygen groups on to carbon nanotubes and changes its
wettability.
Especially for powder samples, here we noted that they are dispersible in water forming a stable colloidal solution. To quantify how stable
is those solutions, zeta potential was measured from nanoparticles
dispersed in water (pH 7.4). The zeta potential ranged from −37.4 to
− 38.6 mV, which means moderate stability behaviour of the colloid
solution [33,48,49]. The GCN-O property of dispersing in water is a
great achievement of this work, because that is an important property
for many biological applications, including the interaction with the T.
foetus.
After this morphological, structural, chemical characterisation of the
GCN-O, now we present the GCN-O interactions with the T. foetus in
TYM, which is the medium wherein the parasitic protozoa are cultivated
[30]. First, Fig. 4(a–d) presents optical images of (a) GCN-O only, (b)
protozoa only and (c & d) conjugate of them dropped onto titanium
substrate. Fig. 4(c & d) shows photographs after 24 h of incubation of
the mixture of (c) 10 μg/ml and (d) 100 μg/ml of GCN-O in TYM medium containing 104 protozoa/ml. There is no doubt that the conjugate
is formed in both concentrations tested. The spatial distribution of
the protozoa in that medium is normally constant as presented in
Fig. 4(b). As one can see, the typical concentration of the protozoa
changes in the presence of the GCN-O. The protozoa seem to go in
GCN-O directions and attaches to its surface.
Fig. 5 presents SEM images of the protozoa and the GCN-O conjugation. One can note the parasite ranges from 5 to 15 μm in size and is
spindle shaped with its ﬂagella and membrane completed fused with
GCN-O. The protozoa and the GCN-O seem to be entangled. We estimated the bond of the conjugate GCN-O2 and T. foetus, measuring the superﬁcial tension of GCN-O, protozoa and a mixture of them in TYM
medium. We veriﬁed that the GCN-O dispersed in TYM has a surface
tension of around 45.45 mN m−1. In TYM medium, the protozoa have
a surface tension of around 48.9 mN m−1. Considering the difference
between these two surface tensions, we semi-quantify the protozoa adherences onto GCN-O in about 3.5 mN m−1, which we consider strong
due to lightweight of both GCN-O and protozoa. The GCN-O property
of adhering to T. foetus is another great achievement of this work. Considering the strong connection between the parasite and the composite,
we can use it for drug carrier. The composite could be mixed with a speciﬁc drug, e.g. estradiol [25,26] and then put in contact with the parasite.
Silva-Filho et al. studied the surface charge of T. foetus and reported
that it is negatively charged, which is favourable to direct adhesion of
parasitic protozoa on cell culture. T. foetus adhesion is inﬂuenced by
Fig. 2. Raman spectra of (a) as-grown MWCNT and (b) GCN-O samples.
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H. Zanin et al. / Materials Science and Engineering C 41 (2014) 65–69
Fig. 3. EDX spectra of the (a) as-grown MWCNT and (b) GCN-O sample; and XPS (c) C1s and (d) O1s ﬁtted spectra recorded from GCN-O sample. Inset (c & d): Table with a percentage of
the total atoms contained in both (c) as-grown MWCNT and (d) GCN-O samples.
the presence of carboxyl groups exposed on the cell surface [50].
Bonilha et al. conﬁrmed the negative surface of T. foetus and showed
that the parasite has strong adhesion to inert substrates such as plastic
and glass maybe due to its oligosaccharide composition [51]. Lobo et
al. [21] showed that according to the thermodynamic theory, the
parasite adhesion is favourable if the work of adhesion is negative. In
this case, the parasite adhesion becomes more thermodynamically
favourable to parasite adhesion when oxygen plasma functioned
VACNT was used.
The oxygen plasma brings negative charge to the carbon material
surface, attaching carboxyl groups on that, which interferes with their
afﬁnity to the negatively charged T. foetus surface. Now the main question remains, if both surfaces of GCN-O and T. foetus are negatively
charged, then why do they form such strong conjugate? There are a
Fig. 4. Photomicrographies showing titanium substrate with dropped solution of (a) GCNO sample in TYM; (b) 104 Tritrichomonas foetus/ml of TYM; and (c) 10 μg/ml and
(d) 100 μg/ml of GCN-O samples in 104 Tritrichomonas foetus/ml of TYM after 24 h of incubation in all cases.
number of possible suggested reasons to explain this, including mainly
(a) GCN-O forms tight helices with T. foetus [52]; (b) the GCN-O and
T. foetus form π–π stacking [53] and (c) by needle-like mechanism,
GCN-O accesses the membrane of the parasite, pierces it and remains
stuck in their [54]. All these hypotheses need to be evaluated with caution, further, only systematic drug carrier study may conﬁrm if this
novel nanomaterial is effective drug carrier and helps with the protozoa
control.
4. Conclusion
We reported a simple, fast and scalable method to produce a GCN-O
composite. First, we prepared MWCNT using CVD furnace and then we
used oxygen plasma etching to exfoliate MWCNT from its tips. Also we
showed that oxygen plasma oxidised this novel material, changing its
hydrophilicity. The GCN-O formed stable colloidal solution in water,
Fig. 5. SEM images of graphene oxide and carbon nanotube oxide (GCN-O2) composite
and Tritrichomonas foetus (Tf) afﬁnity.
H. Zanin et al. / Materials Science and Engineering C 41 (2014) 65–69
which was essential to its application as biomaterial. In several separated experiments, we mixed parasitic protozoa T. foetus with GCN-O and
we noted a strong afﬁnity between them. In this work, we conﬁrmed
the strong adhesion between the composite and the protozoan, as previously reported [21]. The previous studies showed the strong adhesion
of T. foetus to superhydrophylic VACNT surface and we assume that the
adhesion is favourable if the work of adhesion is negative. Herein, our
results showed that the T. foetus can strongly interact with low and
high levels of GCN-O, which could be employed as drug carriers. Although preliminary, these experiments have shown that such GCN-O
hybrid composite may be excellent candidates for a drug carrier, and
more studies should be addressed to better understand this phenomenon. Further, the drug carrier applications of GCN-O are underway in
our group comparing different drugs.
[18]
[19]
[20]
[21]
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Acknowledgements
[33]
The electron microscopy work was performed with a SEM (JEOL
6330) microscope at the LME/LNLS‐Campinas. We also gratefully
acknowledge the Brazilian agencies CNPq (202439/2012-7) and FAPESP
(2011/17877-7, 2011/20345-7) and 2008/06654-4 for ﬁnancial support.
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