materials
Article
High Performances of Artificial Nacre-Like Graphene
Oxide-Carrageenan Bio-Nanocomposite Films
Wenkun Zhu 1,2, *, Tao Chen 2 , Yi Li 1 , Jia Lei 2 , Xin Chen 1 , Weitang Yao 1 and Tao Duan 1, *
1
2
*
State Key Laboratory Cultivation Base for Nonmetal Composites and Functional Materials, Southwest
University of Science and Technology, Mianyang 621010, China; [email protected] (Y.L.);
[email protected] (X.C.); [email protected] (W.Y.)
Engineering Research Center of Biomass Materials, Ministry of Education, Mianyang 621010, China;
[email protected] (T.C.); [email protected] (J.L.)
Correspondence: [email protected] (W.Z.); [email protected] (T.D.);
Tel.: +86-816-608-9883 (W.Z.); +86-816-608-9471 (T.D.)
Academic Editor: Biqiong Chen
Received: 15 February 2017; Accepted: 4 May 2017; Published: 16 May 2017
Abstract: This study was inspired by the unique multi-scale and multi-level ‘brick-and-mortar’
(B&M) structure of nacre layers. We prepared the B&M, environmentally-friendly graphene
oxide-carrageenan (GO-Car) nanocomposite films using the following steps. A natural polyhydroxy
polymer, carrageenan, was absorbed on the surface of monolayer GO nanosheets through
hydrogen-bond interactions. Following this, a GO-Car hybridized film was produced through
a natural drying process. We conducted structural characterization in addition to analyzing
mechanical properties and cytotoxicity of the films. Scanning electron microscope (SEM) and X-ray
diffraction (XRD) analyses showed that the nanocomposite films had a similar morphology and
structure to nacre. Furthermore, the results from Fourier transform infrared spectroscopy (FT-IR),
Raman spectroscopy, X-ray photoelectron spectroscopy (XPS) and Thermogravimetric (TG/DTG)
were used to explain the GO-Car interaction. Analysis from static mechanical testers showed
that GO-Car had enhanced Young’s modulus, maximum tensile strength and breaking elongation
compared to pure GO. The GO-Car nanocomposite films, containing 5% wt. of Car, was able to
reach a tensile strength of 117 MPa. The biocompatibility was demonstrated using a RAW264.7 cell
test, with no significant alteration found in cellular morphology and cytotoxicity. The preparation
process for GO-Car films is simple and requires little time, with GO-Car films also having favorable
biocompatibility and mechanical properties. These advantages make GO-Car nanocomposite films
promising materials in replacing traditional petroleum-based plastics and tissue engineering-oriented
support materials.
Keywords: nacre; graphene oxide; carrageenan; self-assembly; nanocomposite films
1. Introduction
Evolution over hundreds of millions of years has provided natural biomaterials with perfect
structures and functions. Among the numerous natural biomaterials, nacre has received widespread
attention, due to its unique layered structure, high strength and outstanding toughness. The structural
model of nacre has inspired people to prepare light, highly strong and ultra-tough layered
nanocomposites [1–4]. The main components of natural nacre are aragonite-style calcium carbonate
(about 95% wt.) and organic substrates (5% wt.). Nacre has a unique multi-scale, multi-level and
orderly ‘brick-and-mortar’ (B&M) composition structure. Specifically, organic substrates firmly hold
the calcium carbonate sheets together, playing a role like cement. However, this structure can effectively
disperse the pressure imposed on the shell, which is a very favorable mechanical property. The organic
Materials 2017, 10, 536; doi:10.3390/ma10050536
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Materials 2017, 10, 536
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substrates of the structured nanocomposite have the effect of dispersing energy, while the layers of
inorganic calcium carbonate play a key role in enhancing the mechanical properties of nanostructures.
Over the last few years, inspired by the unique multi-scale and multi-level B&M composition
structure in nacre, many studies have reported a series of bio-inspired, highly strong and ultra-tough
composites. These composites have been produced using diverse self-assembly techniques, such
as layer by layer (LBL) deposition [5–9], vacuum filtration-assisted assembly [10–13], freeze-drying
assembly [14–17], interface-assisted self-assembly [1,7] and Langmuir–Blodgett assembly [18–20].
Two-dimensional inorganic assembly units, such as glass flakes, alumina flakes, graphene oxide,
layered double hydroxides, nano-clay and other units, serve as the ‘bricks’, while the high
polymers act as the ‘mortar’. It is important to note that graphite oxide (GO) is an ideal
two-dimensional inorganic assembly unit for preparation of nacre-like materials. Various assembly
techniques have been adopted for the preparation of nacre-like GO-synthetic polymer composite
materials. Using the vacuum-assisted self-assembly technique, Putz et al. prepared polymethyl
methacrylate(PMMA)-graphene oxide materials, which were found to have a mechanical tensile
strength of 180 MPa [21]. Using the self-precipitation assembly technique, Cheng et al. produced
10,12-pentacosadiyn-1-ol(PCDO)-graphene oxide materials with a mechanical tensile strength of
159 MPa [13]. However, few studies focused on the preparation of hybrid nanomaterials using natural
high polymers.
Considering the exhaustion of fossil resources in polymer chemical industries and the
non-degradable waste from organic polymers, one of the most important trends has been to replace
the traditional petroleum-based plastics with natural polymers [8]. Carrageenan (Car), also known
as pelvetia silquosa glue, Carrageen glue or Irish moss glue, is a type of polysaccharide extracted
from red algae in the sea. Car is a mixture of different types of materials and has special physiological
functions, meaning that it has wide applications in the food, medical and chemical industries as well
as in biology fields [22]. At present, reports of Car have mainly focused on the edible blend (such as
starch, konjac glucan-mannan and chitosan). Furthermore, the water resistance and tensile property of
prepared carrageenan food packaging film is poor.
Sijun et al. studied the gel properties of graphene and carrageenan. A schematic diagram has
been proposed to explain the effect of ammonia-functionalized graphene oxide (AGO) on the gelation
of κ-carrageenan. AGO sheets have been shown to attract a number of κ-carrageenan chains through
hydrogen bonding and electrostatic interactions between sulfate groups of κ-carrageenan and amine
groups of AGO [23]. Liu et al. conducted experiments examining GO-carrageenan (GO-Car), with the
resulting GO-carrageenan (GO-Car) composite being further used as a substrate for biomimetic and
cell-mediated mineralization of hydroxyapatite (HA) [22]. However, there is a lack of studies examining
the artificial nacre-like graphene oxide-carrageenan bio-nanocomposite films.
Car has numerous active hydroxyls, thus allowing the preparation of GO-Car nanocomposite films
as a result of the oxygen-bearing functional groups of GO [24]. In the present study, we used inorganic
GO nanosheets to form the ‘bricks’ and carrageenan high-molecular polymers to form the ‘mortar’
for preparation of GO-Car nanocomposite films. Furthermore, we studied the morphology, structure,
mechanical properties and cytotoxicity of GO-Car nanocomposite films. GO-Car nanocomposite
films may have potential applications in replacing traditional petroleum-based plastics and tissue
engineering-oriented support materials.
2. Materials and Methods
2.1. Chemical Reagents
Graphite powder (99%), potassium persulfate (K2 S2 O8 ), phosphorus pentoxide (P2 O5 ), hydrogen
peroxide (H2 O2 , 30%), concentrated sulfuric acid (H2 SO4 , 96%) and hydrochloric acid (HCl) were
provided by Chengdu Kelong Chemical Co., Ltd. (Chengdu, China) and Cheng Du Kelong Chemical
Reagent Company (Chengdu, China). We used de-ionized water in this study. Carrageenan was
Materials 2017, 10, 536
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provided by Mianyang Haomao Konjac Food Co., Ltd (Mianyang, China). All the chemicals listed
above were analytical reagents and none of them underwent purification treatment before use.
2.2. Preparation of GO and Car Solution
GO was prepared using a modified Hummers method as previously described [25,26].
The 1 mg/mL monodisperse and stable GO solution was prepared by putting 0.2 g of graphite oxide
powder into 200 mL water, before being dispersed using ultrasonic waves for 60 min. The 1 mg/mL
carrageenan solution was obtained by adding 0.1 g of carrageenan powder into 100 mL water, before
stirring for 2 h.
2.3. Preparation of GO-Car Nanocomposite Films
The corresponding proportional Car solutions with 0 mL, 2.5 mL, 5 mL and 10 mL were slowly
added to the 50 mL GO solution, with this mixed solution being continuously stirred for 1 h. Following
this, about 40 to 45 mL GO-Car solutions with different mass ratios were poured into four petri dishes
at room temperature to be naturally dried. Finally, 5% GO-Car nanocomposite films, 10% GO-Car
nanocomposite films and 20% GO-Car nanocomposite films were obtained.
2.4. Characterization
To characterize the GO-Car nanocomposite films, scanning electron microscope (SEM) images
were obtained using the Ultra 55 machine (Zeiss, Jena, Germany). The test film was sliced into
10 mm × 100 mm films before SEM scanning. X’Pert PRO (PANalytical, Almelo, The Netherlands)
X-ray diffractometer was used to measure the solid X-ray diffraction (XRD) diagram, using Cu Kα
(λ = 0.154 nm) radiation with 36 kV and 20 mA as the testing voltage and electric current at 4◦ /min.
The scan range 2θ was set at 3◦ –50◦ during testing. Nicolet-5700 (Nicolet, New York, NY, USA) was
used to scan the Fourier Transform Infrared Spectroscopy (FT-IR) spectrogram in a mode of attenuated
total reflectance (ATR), with a wavelength range of 4000–225 cm−1 . In the temperature range of
approximately 40–900 ◦ C, the organic substance and its content in the products was characterized by
Thermogravimetric (TG/DTG) at the heating rate of 20 ◦ C/min. X-ray photoelectron spectroscopy
(XPS) was measured with the monochromatic light of ALK α.h, using a power of 150 W and a beam spot
at 500 µm, which was fixed through a 25-eV energy analyzer. For the surface measurement, the core
level spectrum was measured from the angle of 90◦ . The mechanical properties (tensile strength) of
composite films were measured using static mechanical testers (Instron 5565A, Beijing, China), with
the distance between the two fixtures being 5 mm at a moving speed of 10 mm/min. Films were cut
into 23 mm × 5 mm rectangular films before testing.
2.5. Cytotoxicity Assays
Mouse peritoneal macrophages, RAW264.7 cells, were grown in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% calf serum (HyClone) and 1% antibiotics (100 Units per
mL penicillin and 100 mg/mL streptomycin), before cultured at 37 ◦ C with 5% CO2 . Disinfected
GO-Car nanocomposite films (3 mm × 3 mm) were placed into 24-well plates, with 1 mL DMEM
being added into each well. These films were soaked and moistened for 24 h, with the media then
being removed from plates. Two milliliters of RAW264.7 cells with a concentration of 2 × 106 per mL
and 2 mL of RAW264.7 cell DMEM culture was added to the sample surface in each hole. After this,
RAW264.7 cells with film treatment were cultured at 37 ◦ C with 5% CO2 for 48 h. The inverted
microscope (Nikon ECLIPSE Ti-S, Kanagawa, Japan) was used to observe cell adhesion, cellular
morphology and growth situation, with the MTT assay being used to analyze effects of RAW264.7
on proliferation rates and to evaluate cell cytotoxicity of GO-Car composite films following the
manufacturer’s instructions [9,27].
Materials 2017, 10, 536
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3. Results and Discussion
Figure 1 shows the process designed for preparation of GO-Car composite films. First, we separated
monolayer GO nanosheets from the water solution, before adding carrageenan solution into
the solution of separated GO nanosheets. This method of adding the solution is intended to
facilitate
Car molecules absorbing onto the surface of GO nanosheets through hydrogen-bond
Materials 2017, 10, 536
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interactions. Two-dimensional orientations of remarkable cohesive deposition of Car after being
interactions.
Two-dimensional
remarkable
cohesive hybrid
deposition
of Car
after being
naturally
dried gave
rise to orderlyorientations
nacre-like of
structures
in GO-Car
films.
Compared
with the
naturally
dried
gave
rise
to
orderly
nacre-like
structures
in
GO-Car
hybrid
films.
Compared
layer-by-layer and other assembly techniques, the whole preparation process was simplewith
andthe
required
layer-by-layer and other assembly techniques, the whole preparation process was simple and
less time, which facilitates large-scale production.
required less time, which facilitates large-scale production.
Figure 1. Scheme for fabricating green graphene oxide-carrageenan (GO-Car) composite films.
Figure 1. Scheme for fabricating green graphene oxide-carrageenan (GO-Car) composite films.
Nacre-like GO-Car nanocomposite films were prepared through self-evaporation deposition.
The imagesGO-Car
of prepared
nanocomposite
filmswere
are shown
in Figure
2a. The
slice was soft, with
a
Nacre-like
nanocomposite
films
prepared
through
self-evaporation
deposition.
smooth
Figure
2c–e depicts the
SEM
images
of the
cross-sections
GO-Car
The images
of surface.
prepared
nanocomposite
films
are
shown
in Figure
2a. Theofslice
wasnanocomposite
soft, with a smooth
with2c–e
different
mass
pure GO films in
2b,nanocomposite
there was less space
surface.films
Figure
depicts
theratios.
SEMCompared
images ofwith
the cross-sections
of Figure
GO-Car
films with
between the GO-Car hybridized nanosheets. The dense parallel stacking and highly oriented layered
different mass ratios. Compared with pure GO films in Figure 2b, there was less space between the
structure was quite similar to the B&M structure of nacre. The oxygen-bearing functional groups in
GO-CarGO
hybridized
Thehydroxyls
dense parallel
stacking
and highly
oriented layered
structure was
reacted withnanosheets.
macromolecular
in carrageenan
through
strong hydrogen-bond
interactions.
quite similar
to the B&M
structure of
nacre. The
groupsorientation
in GO reacted
Furthermore,
self-evaporation
deposition
alsooxygen-bearing
led to a strong functional
two-dimensional
and with
macromolecular
hydroxyls
carrageenan
through
hydrogen-bond
interactions.
increased the
adhesive in
action
of Car. Both
factors strong
contributed
to the production
of closely Furthermore,
layered
nacre-like structures,
which
the adhesive
force among orientation
graphene oxide
self-evaporation
deposition
also enhanced
led to a strong
two-dimensional
andnanosheets.
increased the adhesive
action of Car. Both factors contributed to the production of closely layered nacre-like structures,
which enhanced the adhesive force among graphene oxide nanosheets.
To investigate the diffraction properties of GO-Car nanocomposite films, we conducted XRD
analyses for carrageenan, GO and 5% GO-Car nanocomposite films. As shown in Figure 3, Car mainly
existed in an amorphous state, with no obvious characteristic diffraction peaks [28]. Pure graphene
oxide films had a diffraction peak at 2θ = 10.6◦ , corresponding to (002) crystal planes of graphite
reflection with a 0.84 nm interlayer spacing [29]. For 5% GO-Car nanocomposite films, the characteristic
diffraction peak was at 2θ = 8.6◦ , corresponding to (002) crystal planes of graphite reflection with
a 1.14 nm interlayer spacing [30]. The above results may be due to the GO nanosheets having absorbed
Car macromolecules through hydrogen-bond interactions, resulting in a widened interlayer spacing
among graphene oxide nanosheets.
According to the results from the Fourier Transform Infrared Spectroscopy (FT-IR) spectrogram
(Figure 4) of carrageenan and 5% wt. GO-Car hybrid, there were strong interactions between GO and
Car molecules. In the IR spectrum of carrageenan, the absorption peaks at 2927 and 1374 cm−1 were the
stretching vibration and bending vibration of carbohydrate C–H, respectively. The absorption peaks
Figure 2. Morphological images of nacre-like GO-Car nanocomposite films: (a) Images of GO, Car
at 1260, 928 and 846 cm−1 were the characteristic absorption peaks of C–O–C and C–O–S stretching
and GO-Car nanocomposite films. The other parts show the typical SEM images of the fracture surface
vibrations in
carrageenan
molecules. films at different magnifications: (b) Pure GO film; (c) 5% GO-Car;
of GO
and GO-Car nanocomposite
(d) 10% GO-Car and (e) 20% GO-Car.
between the GO-Car hybridized nanosheets. The dense parallel stacking and highly oriented layered
structure was quite similar to the B&M structure of nacre. The oxygen-bearing functional groups in
GO reacted with macromolecular hydroxyls in carrageenan through strong hydrogen-bond interactions.
Furthermore, self-evaporation deposition also led to a strong two-dimensional orientation and
increased
the
action of Car. Both factors contributed to the production of closely layered
Materials
2017,
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536
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nacre-like structures, which enhanced the adhesive force among graphene oxide nanosheets.
Materials 2017, 10, 536
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To investigate the diffraction properties of GO-Car nanocomposite films, we conducted XRD
analyses for carrageenan, GO and 5% GO-Car nanocomposite films. As shown in Figure 3, Car mainly
existed in an amorphous state, with no obvious characteristic diffraction peaks [28]. Pure graphene
oxide films had a diffraction peak at 2θ = 10.6°, corresponding to (002) crystal planes of graphite
reflection with a 0.84 nm interlayer spacing [29]. For 5% GO-Car nanocomposite films, the
characteristic diffraction peak was at 2θ = 8.6°, corresponding to (002) crystal planes of graphite
reflection
with
a 1.14 nm interlayer spacing [30]. The above results may be due to the GO nanosheets
Materials
2017,
10, 536
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having absorbed Car macromolecules through hydrogen-bond interactions, resulting in a widened
To investigate
the diffraction
of GO-Car nanocomposite films, we conducted XRD
interlayer
spacing among
grapheneproperties
oxide nanosheets.
analyses for carrageenan, GO and 5% GO-Car nanocomposite films. As shown in Figure 3, Car mainly
existed in an amorphous state, with no obvious characteristic diffraction peaks [28]. Pure graphene
oxide films had a diffraction peak at 2θ = 10.6°, corresponding to (002) crystal planes of graphite
reflection with a 0.84 nm interlayer spacing [29]. For 5% GO-Car nanocomposite films, the
characteristic diffraction peak was at 2θ = 8.6°, corresponding to (002) crystal planes of graphite
Figure 2. Morphological
images ofspacing
nacre-like
GO-Car
nanocomposite
films:
(a) Images
of
GO,
Car
reflection
a 1.14 nm interlayer
[30].
Thenanocomposite
above
resultsfilms:
may
be
to of
theGO,
GO
nanosheets
Figurewith
2. Morphological
images of nacre-like
GO-Car
(a) due
Images
Car
and
and
GO-Car
nanocomposite
films.
The
other
parts
show
the
typical
SEM
images
of
the
fracture
surface
having
absorbed
Car macromolecules
through
hydrogen-bond
interactions,
in a widened
GO-Car
nanocomposite
films. The other
parts show
the typical SEM
images of theresulting
fracture surface
of
of GO and
GO-Car
nanocomposite
films
atnanosheets.
different magnifications: (b) Pure GO film; (c) 5% GO-Car;
interlayer
spacing
among
graphene
oxide
GO and GO-Car nanocomposite films at different magnifications: (b) Pure GO film; (c) 5% GO-Car;
(d) 10% GO-Car and (e) 20% GO-Car.
(d) 10% GO-Car and (e) 20% GO-Car.
Figure 3. X-ray diffraction (XRD) patterns of GO, Car and 5% GO-Car nanocomposite films.
According to the results from the Fourier Transform Infrared Spectroscopy (FT-IR) spectrogram
(Figure 4) of carrageenan and 5% wt. GO-Car hybrid, there were strong interactions between GO and
Car molecules. In the IR spectrum of carrageenan, the absorption peaks at 2927 and 1374 cm−1 were
the stretching vibration and bending vibration of carbohydrate C–H, respectively. The absorption
peaks at 1260, 928 and 846 cm−1 were the characteristic absorption peaks of C–O–C and C–O–S
stretching vibrations in carrageenan molecules.
Figure
Figure3.3. X-ray
X-ray diffraction
diffraction(XRD)
(XRD)patterns
patternsof
ofGO,
GO,Car
Carand
and5%
5%GO-Car
GO-Carnanocomposite
nanocompositefilms.
films.
1263
1044
1634
2920
2920
3399
928
846
1260
1698
1374
2927
3334
Car
5GO-Car
1263
1634
Wavenumber/ cm-1
1044
4000 3500 3000 2500 2000 1500 1000 500
3334
Transmittance/%
Transmittance/%
According to the results from the Fourier Transform Infrared
CarSpectroscopy (FT-IR) spectrogram
GO-Carinteractions between GO and
(Figure 4) of carrageenan and 5% wt. GO-Car hybrid, there were5
strong
Car molecules. In the IR spectrum of carrageenan, the absorption peaks at 2927 and 1374 cm−1 were
the stretching vibration and bending vibration of carbohydrate C–H, respectively. The absorption
peaks at 1260, 928 and 846 cm−1 were the characteristic absorption peaks of C–O–C and C–O–S
stretching vibrations in carrageenan molecules.
928
846
1698
1374
1260
3399
2927
Figure
Figure4.4. Fourier
Fourier transform
transform infrared
infrared spectroscopy
spectroscopy (FT-IR)
(FT-IR) spectrum
spectrum of
of Car
Car and
and 5%
5% GO-Car
GO-Car
nanocomposite
film.
nanocomposite film.
4000 3500 3000 2500 2000 1500 1000 500
Wavenumber/ cm-1
Materials 2017, 10, 536
Materials 2017, 10, 536
6 of 11
6 of 11
After
GO and
and formed
formed GO-Car
GO-Car hybrid
hybrid nanosheets,
nanosheets, the
the C–O–C
C–O–C
After carrageenan
carrageenan was
was absorbed
absorbed on
on GO
−1
−1
−
1
−
1
absorption
peaks were
wereat
at1044
1044cm
cm and
and 1263
1263 cm
cm , ,while
was at
at 1634
1634 cm
cm−−11..
absorption peaks
while the
the C=C
C=C absorption
absorption peak
peak was
−1
There was
was aa narrowing
narrowing in
in the
the strong
strong absorption
absorption band
band of
of OH
OH at
at 3334
3334 cm
cm−1,, probably
the
There
probably due
due to
to the
hydrogen
bond
between
the
graphene
and
carrageenan
molecules
bringing
these
two
molecules
hydrogen bond between the graphene and carrageenan molecules bringing these two molecules closer
closer
together.
Compared
carrageenan,
there
was
new
FT-IRabsorption
absorptionpeaks
peaks in
in GO-Car,
together.
Compared
with with
purepure
carrageenan,
there
was
nono
new
FT-IR
GO-Car,
suggesting
that
no
chemical
bond
was
formed
between
GO
and
Car.
As
the
water
in
the
GO-Car
suggesting that no chemical bond was formed between GO and Car. As the water in the GO-Car
solution
an increase
increase in
in the
the viscosity
viscosity of
of the
the solution.
solution. Carrageenan
Carrageenan molecules
molecules
solution evaporated,
evaporated, there
there was
was an
gradually
entangled
themselves
into
a
network
structure,
with
the
GO
nanosheets
being
encapsulated
in
gradually entangled themselves into a network structure, with the GO nanosheets being encapsulated
the
GO
layer.
Several
hydrogen
bonds
in
the
original
carrageenan
molecules
were
broken,
with
a
new
in the GO layer. Several hydrogen bonds in the original carrageenan molecules were broken, with a new
strong
interaction being
being formed
formed between
between GO
GO and
and the
the molecules
molecules of
of carrageenan.
carrageenan.
strong interaction
−−1
1 is
The
Raman
spectrums
of
Car
and
GO-Car
are
shown
in
Figure
TheGGband
bandatat1605
1605cm
cm
The Raman spectrums of Car and GO-Car are shown in Figure 5.5.The
is
characteristic
of
graphitic
carbon
layers,
corresponding
to
the
tangential
vibration
of
carbon
atoms,
characteristic of graphitic carbon layers, corresponding to the tangential vibration of carbon atoms,
−1
while the
carbon
[31].
The
intensity
ratio
of of
thethe
D
while
the D
D band
band at
at 1338
1338 cm
cm−1indicates
indicatesa adefective
defectivegraphitic
graphitic
carbon
[31].
The
intensity
ratio
band
and
G
band
(ID/IG)
was
1.02.
The
D
band
and
G
band
was
clearly
observed
in
the
GO-Car
D band and G band (ID/IG) was 1.02. The D band and G band was clearly observed in the GO-Car
nanocomposite
films, suggesting
suggesting the
the existence
existence of
of carbon
carbon in
in the
the composite
composite [32].
[32].
nanocomposite films,
Car
5GO-Car
500
1000
1605
Itensity(a.u)
1338
D bond
G bond
1500
2000
2500
Raman Shift/cm-1
3000
Figure 5. Raman
Raman spectrums of Car and 5% GO-Car nanocomposite films.
XPS
wasused
usedinin
order
to further
explore
the interaction
between
Car.
Inspectrum,
the XPS
XPS was
order
to further
explore
the interaction
between
GO andGO
Car.and
In the
XPS
spectrum,
the
position
of
each
line
corresponds
to
the
binding
energy
of
the
electrons.
The
XPS
the position of each line corresponds to the binding energy of the electrons. The XPS spectra spectra
test for
test
Car
withofa C1s
coreisofshown
C1s is in
shown
in6b.
Figure
C1s
eV belonged
C–H,the
while
Car for
with
a core
Figure
The 6b.
C1sThe
peak
at peak
284.7 at
eV284.7
belonged
to C–H,to
while
C1s
the
C1s
peak
at
285.8
eV
belonged
to
C–N.
The
peak
height
of
C–H
was
found
to
be
significantly
peak at 285.8 eV belonged to C–N. The peak height of C–H was found to be significantly higher than
higher
than
the peak
with the
elements
in CarC–H,
beingfollowed
mainly by
C–H,
followed
byspectra
C–N. The
the peak
of C–N,
with of
theC–N,
C elements
in C
Car
being mainly
C–N.
The XPS
test
XPS
spectrawith
test for
GO-Car
a coreinofFigure
C1s is6d.
shown
inbinding
Figure 6d.
The Cwere
binding
were
for GO-Car
a core
of C1swith
is shown
The C
energies
284.7energies
and 286.3
eV,
284.7
and 286.3
eV, respectively,
belonged
to C–H andwith
C=O.Figure
Compared
with
Figure
themain
C–N
respectively,
which
belonged to which
C–H and
C=O. Compared
6d, the
C–N
bond6d,
is the
bond
is the main component in the C1s of carrageenan (Figure 6b), because the carrageenan is a
component in the C1s of carrageenan (Figure 6b), because the carrageenan is a galactose formed by two
galactose formed by two galactoses. In comparison, C=O occupies the main position in GO-Car
galactoses. In comparison, C=O occupies the main position in GO-Car (Figure 6d), which may be the
(Figure 6d), which may be the result of graphene oxide having played a certain role in promotion.
result of graphene oxide having played a certain role in promotion. There was no obvious change in
There was no obvious change in the intensity of each peak, suggesting that there was no new chemical
the intensity of each peak, suggesting that there was no new chemical bond formation during physical
bond formation during physical mixing. It was thought that physical mixing led to the formation of
mixing. It was thought that physical mixing led to the formation of new hydrogen bonds.
new hydrogen bonds.
It can be seen from Figure 7 that the thermal decomposition of Car film began at 65 ◦ C, while the
thermal decomposition of the GO-Car nanocomposite films began at 72 ◦ C. The thermal stability of
GO-Car nanocomposite films was better than the pure Car film. At 100 ◦ C, there was no significant
degradation of weight loss, which might be due to the loss of water molecules in the matrix. It can be
seen from Figure 7 that the thermal decomposition occurred in the range of 100–350 ◦ C. There was
a loss of most of the oxygen-containing functional groups, hastening the weight decline. In GO-Car
nanocomposite films, the oxygen-containing functional groups decomposed into CO and CO2 [31].
Materials 2017, 10, 536
7 of 11
◦
When
the
temperature
exceeded 200 ◦ C, the polymer chain of carrageenan was destroyed. After 500
Materials
2017,
10, 536
7 of C,
11
the thermal weight loss was very slow, with the thermal decomposition gradually becoming stabilized.
Figure 6. X-ray photoelectron spectroscopy (XPS) spectra of (a) Car nanocomposite films; (b) Car
nanocomposite film with a core of C1s; (c) 5% GO-Car nanocomposite films and (d) 5% GO-Car
nanocomposite film with a core of C1s.
It can be seen from Figure 7 that the thermal decomposition of Car film began at 65 °C, while the
thermal decomposition of the GO-Car nanocomposite films began at 72 °C. The thermal stability of
GO-Car nanocomposite films was better than the pure Car film. At 100 °C, there was no significant
degradation of weight loss, which might be due to the loss of water molecules in the matrix. It can be
seen from Figure 7 that the thermal decomposition occurred in the range of 100–350 °C. There was a
loss of most of the oxygen-containing functional groups, hastening the weight decline. In GO-Car
nanocomposite films, the oxygen-containing functional groups decomposed into CO and CO2 [31].
When
the temperature
exceeded spectroscopy
200 °C, the polymer
chain of
was destroyed.
After
Figure
6.
(a) Car nanocomposite
films; (b)
Car500 °C,
6. X-ray photoelectron
(XPS) spectra
of carrageenan
nanocomposite
the thermal
weightfilm
losswith
was very
with
the
thermal
decomposition
gradually
becoming
stabilized.
nanocomposite
a coreslow,
of
C1s;
(c)
5%
GO-Car
nanocomposite
films
and
(d)
5%
GO-Car
of C1s;
5% GO-Car nanocomposite
GO-Car
nanocomposite
nanocomposite film
film with
with aa core
core of
of C1s.
C1s.
Weight (%)
It can be seen from Figure100
7 that the thermal decomposition
Carof Car film began at 65 °C, while the
5GO-Car
thermal decomposition of the GO-Car
nanocomposite films began
at 72 °C. The thermal stability of
90
GO-Car nanocomposite films was better than the pure Car film. At 100 °C, there was no significant
80
degradation of weight loss, which might be due to the loss of water molecules in the matrix. It can be
70
seen from Figure 7 that the thermal
decomposition occurred in the range of 100–350 °C. There was a
loss of most of the oxygen-containing
functional groups, hastening the weight decline. In GO-Car
60
nanocomposite films, the oxygen-containing
functional groups decomposed into CO and CO2 [31].
50
When the temperature exceeded 200 °C, the polymer chain of carrageenan was destroyed. After 500 °C,
40
the thermal weight loss was very slow, with the thermal decomposition gradually becoming stabilized.
30
100
0
100
200
300
400
500
Temperature (°C)
600
700
Car
Weight (%)
5GO-Car
Figure
(TG/DTG)
5%
90
Figure7.7.Thermogravimetric
Thermogravimetric
(TG/DTG)spectra
spectraofofCar
Carand
and
5%GO-Car
GO-Carnanocomposite
nanocompositefilm.
film.
80
In order to demonstrate the enhanced mechanical properties resulted from the nacre-like micro
70
structure, we characterized the tensile strengths of GO-Car nanocomposite films with different mass
60 as the control group (Figure 8). For the 5% GO-Car nanocomposite
ratios, using pure graphene oxide
films, the maximum tensile strength
was 117 MPa and the breaking elongation was 7.82%, meaning
50
that it was 190.90% and 191.79% higher than the corresponding values of pure GO. For the 10% GO-Car
40
nanocomposite films, the maximum Young’s modulus was 21.87 GPa, which was 24.26% higher than
30
that of Car. These significant improvements
can be
0
100 200
300attributed
400 500to the
600 strong
700 hydrogen-bond interactions
Temperature (°C)
Figure 7. Thermogravimetric (TG/DTG) spectra of Car and 5% GO-Car nanocomposite film.
structure,we
wecharacterized
characterizedthe
thetensile
tensilestrengths
strengthsofofGO-Car
GO-Carnanocomposite
nanocompositefilms
filmswith
withdifferent
differentmass
mass
structure,
ratios,using
usingpure
puregraphene
grapheneoxide
oxideasasthe
thecontrol
controlgroup
group(Figure
(Figure8).8).For
Forthe
the5%
5%GO-Car
GO-Carnanocomposite
nanocomposite
ratios,
films,
the
maximum
tensile
strength
was
117
MPa
and
the
breaking
elongation
was
7.82%,
meaning
films, the maximum tensile strength was 117 MPa and the breaking elongation was 7.82%, meaning
thatit itwas
was190.90%
190.90%and
and191.79%
191.79%higher
higherthan
thanthe
thecorresponding
correspondingvalues
valuesofofpure
pureGO.
GO.For
Forthe
the10%
10%GO-Car
GO-Car
that
nanocomposite
films,
the
maximum
Young’s
modulus
was
21.87
GPa,
which
was
24.26%
higher
Materials
2017,
10,
536
ofthan
11
nanocomposite films, the maximum Young’s modulus was 21.87 GPa, which was 24.26% higher8than
that
of
Car.
These
significant
improvements
can
be
attributed
to
the
strong
hydrogen-bond
interactions
that of Car. These significant improvements can be attributed to the strong hydrogen-bond interactions
betweenGO
GOnanosheets
nanosheetsand
and Carmacromolecules
macromolecules inaddition
addition tothe
thelayered
layeredstructure,
structure,which
whichwas
was
between
between
GO
nanosheets
and Car
Car macromolecules in
in addition to
to the layered
structure, which
was
similar
to
natural
nacre.
similar
nacre.
similar to
to natural
natural nacre.
Tensile strength (MPa)
Tensile strength (MPa)
125
125
100
100
Car
Car
5GO-Car
5GO-Car
10GO-Car
10GO-Car
75
75
50
50
25
25
0 00
0
22
44
66
Strain(%)
(%)
Strain
88
10
10
Figure8.8.Typical
Typicalstress-strain
stress-straincurves
curves ofCar
Car andGO-Car
GO-Car nanocomposite
films.
Figure
Figure
8. Typical stress-strain
curves of
of Car and
and GO-Car nanocomposite
nanocomposite films.
films.
Comparedwith
withthe
thepure
pure Car films, there was a dramatic increase in the stability of the hybrid
Compared
Car films, there was a dramatic increase in the stability of the hybrid
film
in
the
wet
environment.
As showninin Figure 9, the pure Car nanocompositefilms
films apparently
film in the wet environment. As
shown Figure 9, the pure Car nanocomposite
apparently
degraded
after
being
immersed
in
water
for
seven
days.
The
film
was
broken
into
many
small
pieces.
degraded after being immersed in water for seven days. The
The film
film was
was broken
broken into
into many
many small
small pieces.
pieces.
However,for
forthe
theGO-Car
GO-Carnanocomposite
nanocompositefilms,
films,there
therewas
wasnegligible
negligibledegradation
degradationand
and thefilm
film was
However,
However, for
the GO-Car
nanocomposite films,
there was
negligible degradation
and the the
film was was
still
still
maintained
with
initial
robustness.
still
maintained
with
initial
robustness.
maintained with initial robustness.
Figure 9. Photographs of different films being immersed in distilled water for different amounts of
Figure 9. Photographs of different films being immersed in distilled water for different amounts of
Figure
9. Photographs
different
being
immersed in distilled
forCar
different
amounts
of
time: (a)
Car film for 5ofmin;
(b) 5%films
GO-Car
nanocomposite
film for 5water
min; (c)
film for
7 days and
time: (a) Car film for 5 min; (b) 5% GO-Car nanocomposite film for 5 min; (c) Car film for 7 days and
time:
(a)
Car
film
for
5
min;
(b)
5%
GO-Car
nanocomposite
film
for
5
min;
(c)
Car
film
for
7
days
and
(d) 5% GO-Car nanocomposite film for 7 days.
(d) 5%
5% GO-Car
GO-Car nanocomposite
nanocomposite film
film for
for 77 days.
days.
(d)
Thecytotoxicity
cytotoxicityofofGO-Car
GO-Carnanocomposites
nanocompositeswas
wasevaluated
evaluatedthrough
throughgrowing
growingRAW264.7
RAW264.7cell
cellonon
The
The
cytotoxicity
ofFigure
GO-Car
nanocomposites
was evaluated
through
growing RAW264.7
cell
on
the
the
films
for
48
h.
10
depicts
the
influences
of
GO-Car
nanocomposite
films
on
cellular
the films for 48 h. Figure 10 depicts the influences of GO-Car nanocomposite films on cellular
films
for 48 h. Figure
10proliferation,
depicts the influences
GO-Car microscope
nanocomposite
filmsECLIPSE
on cellular
morphology
morphology
andcell
cell
usingthe
theof
inverted
(Nikon
Ti-S,
Kanagawa,
morphology
and
proliferation, using
inverted
microscope (Nikon
ECLIPSE Ti-S,
Kanagawa,
and cell proliferation, using the inverted microscope (Nikon ECLIPSE Ti-S, Kanagawa, Japan) and MTT
colorimetric assay. Cells adhered on the surface of nanocomposite films had clear outlines, with the
oval shape being similar to the control group tissue culture polystyrene (TCPS). These results indicated
that GO-Car nanocomposite films did not affect cellular morphology and cell proliferation. Therefore,
the nanocomposite films with outstanding cell biocompatibility might have a promising application in
the field of tissue engineering-oriented support materials.
Japan) and MTT colorimetric assay. Cells adhered on the surface of nanocomposite films had clear
outlines, with the oval shape being similar to the control group tissue culture polystyrene (TCPS).
These results indicated that GO-Car nanocomposite films did not affect cellular morphology and cell
proliferation. Therefore, the nanocomposite films with outstanding cell biocompatibility might have
2017, 10,
536
9 of 11
aMaterials
promising
application
in the field of tissue engineering-oriented support materials.
Figure 10. (a,b) Phase contrast and fluorescence images of human umbilical vein endothelial cells
Figure 10. (a,b) Phase contrast and fluorescence images of human umbilical vein endothelial cells
(HUVECs) grown on tissue culture polystyrene (TCPS) plates and a GO-Car nanocomposite films
(HUVECs) grown on tissue culture polystyrene (TCPS) plates and a GO-Car nanocomposite films
coated surface for 48 h; (c) RAW264.7 Cell viability measured by MTT assay after being cultured for 0,
coated surface for 48 h; (c) RAW264.7 Cell viability measured by MTT assay after being cultured for
24, 48 and 72 h.
0, 24, 48 and 72 h.
4. Conclusions
Conclusions
4.
Inspired by
by nacre,
nacre, our
our study
study used
used inorganic
inorganic GO
GO nanosheets
nanosheets as
as the
the ‘bricks’
‘bricks’ and
and carrageenan
carrageenan
Inspired
(natural polymeric
polymeric compound)
compound) as
as the
the ‘mortar’
‘mortar’ to
to mimic
mimic the
the layered
layered structure
structure of
of nacre
nacre for
for preparing
preparing
(natural
the
GO-Car
nanocomposite
films.
The
results
from
SEM,
FT-IR,
Raman,
XRD,
TG/DTG,
XPS,
the GO-Car nanocomposite films. The results from SEM, FT-IR, Raman, XRD, TG/DTG, XPS, Raman
Raman
and
the
Instron
tensile
tester
indicated
that
carrageenan
molecules
can
closely
bond
with
and the Instron tensile tester indicated that carrageenan molecules can closely bond with GO
GO nanosheets
through
hydrogen-bond
interactions
and
form nacre-like
nanocomposite
films.
nanosheets
through
hydrogen-bond
interactions
and form
nacre-like
nanocomposite
films. Young’s
Young’s
modulus,
maximum
tensile
strength
and
breaking
elongation
were
significantly
improved
in
modulus, maximum tensile strength and breaking elongation were significantly improved in GO-CAR
GO-CAR
nanosheets
when
compared
with
pure
GO
films.
Furthermore,
such
films
have
favorable
nanosheets when compared with pure GO films. Furthermore, such films have favorable
biocompatibility, indicating
application
in replacing
the traditional
petroleum-based
plastics
biocompatibility,
indicatinga potential
a potential
application
in replacing
the traditional
petroleum-based
and
tissue
engineering-oriented
support
materials.
plastics and tissue engineering-oriented support materials.
Acknowledgments: This work was financially supported by Sichuan Province Science and technology
Acknowledgments:
This work was
financially
supported
by SichuanofProvince
Science
andand
technology
Pillar
Pillar Program (No. 2014GZ0185),
National
Natural
Science Foundation
China (No.
21601147
21406182),
Key
Program
(No.
2014GZ0185),
National
Natural
Science
Foundation
of
China
(No.
21601147
and
21406182),
Key
research and development project of science and technology department of Sichuan province (No. 2017GZ0342),
research
development
project of
science&and
technology
department
of(No.
Sichuan
province (No.
Projects and
in the
Sichuan Province
Science
Technology
Pillar
Program
2016GZ0259
and 2017GZ0342),
2016GZ0277),
Plan Projects
Mianyang
Science
and Technology
(No.Pillar
15zd2110
and (No.
15zd2102).
Projects
in theofSichuan
Province
Science
& Technology
Program
2016GZ0259 and 2016GZ0277), Plan
Projects
of Mianyang Science
andZhu
Technology
(No. 15zd2110
and
15zd2102).
Wenkun
and Tao Duan
conceived
and
designed the study; Tao Chen and Xin Chen
Author Contributions:
performed the experiments; Yi Li, Jia Lei and Weitang Yao analyzed the data; Wenkun Zhu and Tao Chen wrote
the majority of the paper and all authors reviewed and approved the final version.
Conflicts of Interest: The authors declare no conflict of interest.
Materials 2017, 10, 536
10 of 11
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