Accepted Manuscript

Title: Covalently functionalized graphene sheets with

biocompatible natural amino acids
Author: Shadpour Mallakpour Amir Abdolmaleki Sedigheh
Borandeh
PII:
DOI:
Reference:

S0169-4332(14)00834-4
http://dx.doi.org/doi:10.1016/j.apsusc.2014.04.070
APSUSC 27670

To appear in:

APSUSC

Received date:
Revised date:
Accepted date:

11-12-2013
7-4-2014
9-4-2014

Please cite this article as: S. Mallakpour, A. Abdolmaleki, S. Borandeh, Covalently

functionalized graphene sheets with biocompatible natural amino acids, Applied Surface
Science (2014), http://dx.doi.org/10.1016/j.apsusc.2014.04.070
This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof
before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that
apply to the journal pertain.

Revised
Covalently functionalized graphene sheets with biocompatible natural

Shadpour Mallakpour a,b,, Amir Abdolmaleki a,b,, Sedigheh Borandeh a

Organic Polymer Chemistry Research Laboratory, Department of Chemistry, Isfahan

University of Technology, Isfahan, 84156-83111, I. R. Iran.

Nanotechnology and Advanced Materials Institute, Isfahan University of Technology,

us

cr

ip
t

amino acids

Ac
ce
p

te

an

Isfahan, 84156-83111, I. R. Iran.

Corresponding author: Tel.; +98-311-391-3249; Fax: +98 3113912350.

E-mail addresses: abdolmaleki@cc.iut.ac.ir, amirabdolmaleki@yahoo.com (A. Abdolmaleki);
mallak@cc.iut.ac.ir, mallak777@yahoo.com, mallakpour84@alumni.ufl.edu (S. Mallakpour)

Page 1 of 28

Abstract
Graphene sheets were covalently functionalized with aromatic-aliphatic amino acids,
(phenylalanine and tyrosine), and aliphatic amino acids (alanine, isoleucine, leucine,
methionine and valine) by simple and green procedure. For this aim, at first natural graphite

ip
t

was converted into graphene oxide (GO) through strong oxidation procedure; then, based on
the surface-exposed epoxy and carboxylic acid groups in GO solid, its surface modification

cr

with naturally occurring amino acids, occurred easily throughout the corresponding
nucleophilic substitution and condensation reactions. Amino acid functionalized graphene

us

demonstrates stable dispersion in water and common organic solvents. Fourier transform
infrared, Raman and X-ray photoelectron spectroscopies, X-ray diffraction, field emission
scanning electron microscopy and transmission electron microscopy were used to investigate

an

the nanostructures and properties of prepared materials. Each amino acid has different
considerable effects on the structure and morphology of the pure graphite, from increasing
the layer spacing to layer scrolling, based on their structures, functional groups and chain

length. In addition, therogravimetric analysis was used for demonstrating a successful

grafting of amino acid molecules to the surface of graphene.

Ac
ce
p

oxide.

te

Keywords: Graphite sheet; Amino acid functionalization; Graphene scrolling; Graphene

Page 2 of 28

1. Introduction
In nanoscience field, carbon-based nanomaterials play an important role and have
attracted the scientific community since their discovery [1]. Among these materials, graphene
is one of the most exciting materials which has fascinated great interest in the past several

ip
t

years owing to its potential applications. Graphene, a single layer of sp2-bonded carbon
atoms, is a two-dimension (2D) honeycomb nanostructure [2-6]. The outstanding properties

cr

of graphene as well as their functionalized forms render it an ideal candidate in a wide range
of applications; including composite materials, gas sensors, transparent electrodes and

us

transistors [7-11]. Due to characteristic structures of carbon-based nanomaterials, they can

interact with wide variety of organic molecules by covalent or noncovalent forces (hydrogen
bonding, stacking, electrostatic forces, van der Waals forces and hydrophobic

an

interactions) [12-15].

However, the existence of high stacking and van der Waals forces due to

attraction between adjacent layers facilitates graphene forming, irreversible aggregation or

even restacking to graphite which greatly limits the applications of graphene in several areas,
as well as fabrication of graphene based polymer nanocomposites, biosensors, drug delivery

systems, solar cells, nanomechanical and transistor devices [13,16-21]. Along with numerous

te

approaches that developed to address this obstacle, the most reliable techniques are
functionalization of graphene [22-24]. Several methods have been used for the modification

Ac
ce
p

of graphene which can be divided into two common categories: covalent and non-covalent
functionalization. Utilizing of graphene oxide (GO) as a precursor, has received great
attention of a significant number of researchers these days. GO is an oxygen-containing
graphene derivative with partial breakage of sp2sp2 bonds into sp3sp3 bonds for inserting
some pendent groups like; hydroxy, epoxy, and carboxylic acid. These functional groups
facilitate the interaction between the host materials and GO, also they lead to good dispersion
of GO in aqueous solutions thanks to the hydrophilic nature of surface functionalities [25-29].
On the other hand, despite GO sheets readily swell and disperse in aqueous media but
they cannot be readily dispersed in most common organic solvents [3,12,30]. So, in order to
increase the dispersibility of graphene in various solvents (aqueous and organic), further
functionalization (amidation, esterification, sulfonation and etc) is needed [13,15,31-33].
Inserting different kinds of functional groups onto graphene layers leads to various changes
on graphene structure such as increasing the interlayer spacing or layer scrolling that possess
3

Page 3 of 28

the structure distinct from graphite and multi-walled carbon nanotubes [34-37]. In addition,
nowadays, the biological applications of graphene have also been interested [38-42]. Aiming
this goal, biocompatible equipments may be rewarded while using multi-functional natural
metabolites such as amino acids. These materials are environmentally friendly and naturally

ip
t

occurring compounds, that make graphene a good candidate for biological activities [43-50].
In this context, bio-functionalized graphene sheets with aromatic-aliphatic and

cr

aliphatic amino acids have been prepared through easy and green procedure. For this propose,
at first GO was synthesized using the simplified Hummers method [51], then amino acid

us

functionalized graphene materials were synthesized by condensation and nucleophilic

addition reactions between NH2 groups of amino acid and carboxylic acid or epoxy groups
on the GO sheets. All synthesized amino acid functionalized graphene were characterized by

an

several techniques including Fourier transform infrared spectroscopy (FT-IR), X-ray

diffraction (XRD) and one of them was structurally characterized by Raman spectroscopy
and X-ray photoelectron spectroscopy (XPS). The thermal properties of graphite, GO and

functionalized graphene materials were examined through thermogravimetric analysis

(TGA). Moreover their morphology was investigated by field emission scanning electron

microscopy (FE-SEM). Furthermore, two of them were examined as representative by

Ac
ce
p

2. Experimental

te

transmission electron microscopy (TEM) analysis.

2.1. Materials

Natural graphite powder (diameter 510 m, thickness 4-20 nm, layers < 30 and

purity >99.5 wt%), was purchased from Neutrino Co. (Iran). Other chemicals used in this
study were obtained from Fluka Chemical Co. (Switzerland) and Merck Chemical Co.
(Germany) and were used without further purification. Sulfuric acid (H2SO4 98%), hydrogen
peroxide (H2O2), and hydrochloric acid (HCl), from Merck were used for the synthesis of
mediators.

2.2. Instrumentation
FT-IR spectra of the composites were recorded with a Jasco-680 (Japan) spectrometer
at 4 cm1 resolution and they were scanned at wavenumber range 4004000 cm1. XRD was
used to characterize the crystalline structure of the composites. XRD patterns were collected
using a Bruker, D8 Advanced diffractometer with a copper target at the wave length of
4

Page 4 of 28

CuK = 1.5406 and a tube voltage of 40 kV and tube current of 35 mA, in the range of 5
100 at the speed of 0.05 /min. Raman spectroscopy was recorded from 500 to 3500 cm-1 on
a Almega Thermo Nicolet Dispersive Raman Spectrometer using a Nd:YLF laser source
operating at wavelength of 532 nm. X-ray photoelectron spectroscopy (XPS) was utilized to

ip
t

investigate chemical states variations of the functionalized graphene using twin anode
XR3E2 X-ray source system operating at a vacuum by X-ray 8025-BesTec spectrometer. The
XPS peaks were deconvoluted by using Voigt function which is combination of Lorentzian

cr

and Gaussian. Thermogravimetric analysis (TGA) is performed with a STA503 win TA

(Bahr-Thermoanalyse GmbH, Hllhorst, Germany) at the heating rate of 10C/min from 25
C to 800 C under nitrogen atmosphere. The morphology of several amino acid

us

functionalized graphene were observed using FE-SEM (HITACHI S-4160, Japan). TEM

an

image was obtained using Philips CM 120 operated (Netherlands) at voltage of 150 kV.
2.3. Synthesis of graphene oxide

Graphene oxide (GO) was exfoliated compared to natural graphite through Hummers
technique [51]. Graphite powder (0.5 g) was poured into cold (0 C) solution of concentrated
H2SO4 (12 mL) and NaNO3 (0.25 g). KMnO4 (1.5 g) was gradually added with stirring and

cooling, so that the temperature of the mixture kept below 20 C. The mixture was then

te

stirred at 35 C for 30 min and as the reaction progressed, the mixture gradually became
pasty, and the color turned from black into light brownish. Then, distilled water (25 mL) was

Ac
ce
p

added and the temperature was raised to 98 C and maintained at this temperature for 15
minutes. The reaction was terminated by addition of a large amount of distilled water (70
mL) followed by treated with 30% H2O2 (2 mL) and the mixture changed into brilliant yellow
color. The mixture was filtered and washed with distilled water and 10% HCl solution in
order to remove metal ions. The obtained graphite oxide powder was dispersed in deionized
water. The resulting yellow brownish suspension was centrifuged at 3000 rpm for 1 h to
eliminate unexfoliated graphitic plates. Finally, an aqueous suspension containing GO sheets
was obtained through exfoliation of the filtered graphite oxide suspension through its
sonication at frequency of 2.25 104 Hz and power of 100 W for 1 h. Finally the GO powder
was dried at 60 C.
2.4. Functionalization of GO with different aromatic-aliphatic and aliphatic amino acids
GO powder (0.1 g) was dispersed in distilled water (10 mL) and different amino acids
(0.3 g) (phenylalanine, tyrosine, alanine, leucine, isoleucine, methionine and valine) and an
5

Page 5 of 28

equimolar amount of NaOH in distilled water (10 mL) were added. The mixture was stirred
for 24 h at room temperature. At the end of the reaction the colloidal dispersion was treated
with ethanol, and the resulting precipitate was centrifuged, washed well with H2O/EtOH
mixture and finally dried at 60 C.

ip
t

3. Results and discussion

3.1. Preparation of amino acid functionalized graphene

cr

Amino acids were chosen to fabricate bio-functionalized graphene. At first, chemical

us

oxidation technique fabricates GO by introducing carboxylic acid, hydroxy and epoxy

functional groups between carbon layers of graphene through strong oxidation process (Fig.
1). By means of these functional groups, the van der Waals bond between the carbon layers

an

reduces which cause graphene to peel off layer by layer. The resulting GO can be further
functionalized with numerous compounds. Amino acids are cheap and environmentally
friendly, therefore, they are appropriate nucleophilic reagents. As shown in Fig. 1,

nucleophilic and condensation reactions occurred between the amine groups of amino acids
with epoxy and carboxylic acid groups of the GOs surface. The surface and morphology of

Fig. 1.

Ac
ce
p

te

and TEM methods.

the prepared materials were analyzed by FT-IR, Raman, XPS spectroscopy, XRD, FE-SEM,

3.3. Amino acid functionalized graphene Characterization

3.3.1. FT-IR analysis

FT-IR measurement was employed to investigate the bonding interactions in natural

graphite before and after the oxidation and functionalization processes. In the GO spectrum
(Fig. 2) comparing to the neat graphite, the presence of a peak at 1046 cm-1 attributed to the
C-O bond, confirming the presence of oxide functional groups after the oxidation process. In
addition, the presence of different types of functionalities in GO cause the appearance of
absorption band centered at 3430 cm-1, which is attributed to the O-H stretching bands of
hydroxy and carboxylic acid moieties. Aliphatic sp3 C-H stretching around 2922 cm-1 and the
C=O stretching groups of carboxylic acids at 1719 cm-1, that prove the oxidation process,
while no significant peak was found in the pure graphite.

Page 6 of 28

In different amino acid functionalized graphene spectra, the existence of C=O

stretching groups of amide and carboxylate salts at the range of 1615-1630 cm-1 and the peak
attributed to the OH and NH stretching groups at 3450 cm-1 confirm the covalent
functionalization of the neat graphite by amino acids molecules. In addition, for better

ip
t

comparisons, the FT-IR spectra of phenylalanine and tyrosine functionalized garaphene were
compared with corresponding amino acids. Appearance of amino acid absorption peaks in the

cr

FT-IR spectrum of functionalized garaphene, confirmed successful functionalization process.

Fig. 2.

us

3.3.2. Raman spectroscopy

Raman spectroscopy was used as a powerful technique for obtaining an indication of

an

the structure and quality of carbon materials. Therefore, Raman spectroscopy was utilized to
examine the carbon structure of the GO and phenylalanine functionalized graphene as shown
in Fig. 3. Generally, the well-known and main characteristics of graphite Raman spectrum are

the D and G bands. The G band (strong band) at 1575 cm1 is assigned to the first order
scattering of the E2g phonon from sp2 carbon atoms and the D band (very weak band) at 1355

cm1 is attributed to a breathing mode of -point photons of A1g symmetry which is related to
the local sp3 disorder bands formation through oxidation process especially the ones located

te

at the edges of graphite sheets [41,52,53]. So the intensity ratio of the D and G band (ID/IG) is
a useful parameter for determining the sp2 domain size of a carbon structure containing sp3

Ac
ce
p

and sp2 bonds. Compared with pure graphite, GO exhibited the G band at 1593 cm1 and the
D band at 1352 cm1 (Fig. 3a). In the GO Raman spectrum, the intensity of the D band was
increased compared with that of graphite but the G band is still prominent and the ID/IG (an
indication of sp3/sp2 carbon ratio) for GO is 0.90. According to the Fig. 3b, it is found that by
functionalization of GO with phenylalanine, the G and D bands are shifted to 1594 and 1349
cm1, respectively and the D band becomes more prominent. Higher ID/IG ratio of
functionalized graphene (1.06) compared to GO (0.90), approve the introduction of sp3
defects after functionalization of GO with phenylalanine.
Furthermore, Raman spectra of carbon materials possess a famous feature (2D band)
which is sensitive to stacking of graphene sheets. Based on 2D band shape, intensity and
position, the formation and the layer numbers of graphene sheets can be comprehended [5456]. According to the Raman spectrum of phenylalanine functionalized graphene, a broader

Page 7 of 28

and symmetrical 2D band in the 25003200 cm-1 region, proves that bi-layer graphene sheets
with less than five layers were formed by functionalization of GO [56-60].
Fig. 3.

ip
t

3.3.3. XRD studies

Fig. 4 shows the XRD patterns of graphite and GO. The noticeable peak of graphite
(002) at 26.6 has an interplanar distance, d002, of 3.26 . This signifies that graphite is a

cr

highly oriented carbon material. Whereas, the XRD pattern of GO shows a strong and sharp

us

peak centered at 11.6 which corresponds to the layer-to-layer distance of 7.7 [61,62]. This
outstanding shift signifies that the abundant functional groups are involved and increased the
interlayer spacing of GO due to the intercalation and the bonding effect of O-containing

an

functional groups.

Fig. 4.

Figs. 5 and 6 show the XRD patterns of variety of aromatic-aliphatic and aliphatic
amino acid functionalized graphene. As it can be seen, there are noticeable differences within

the XRD patterns of functionalized GO with aromatic-aliphatic and aliphatic amino acids. In
contrast to aliphatic amino acids, the insertion of aromatic ones, especially tyrosine,

te

decreasing the layer-to-layer distance and increasing the 2 position was observed; in fact,
after functionalization of GO with tyrosine molecule, the peak shifted back to the original

Ac
ce
p

002 peak at 26. This XRD pattern reveals that tyrosine based graphene demonstrates a
particular scrolled structure between graphene and multi-walled carbon nanotubes that comes
from rolling single layer of graphene. Along with numerous researches, a theoretical study on
the interaction of aromatic amino acids with graphene has been examined by Rajesh et al.
within linear combination of atomic orbitals-molecular orbital approach [63]. As they
revealed, the aromatic rings of the amino acids prefer to orient in parallel with the benzene
rings plane of graphene, which is characteristics of the - stacking interactions. They have
been also proved that the binding strength or interaction energy between the aromatic ring of
tyrosine and graphene is higher than the one between the aromatic ring of phenylalanine and
graphene by theoretical calculation.
While the mechanism for this unpredicted phenomenon is unknown, we suspect that
the shift in the interlayer spacing has been attributed to the nature of tyrosine molecules to
adopt a flat orientation in the interlayer zone of GO, presumably due to the - stacking
8

Page 8 of 28

interaction between the aromatic ring of tyrosine and graphene and also H-bonding
interactions between the phenolic OH groups of guest molecules and O-containing groups of
GOs layers. So, based on above explanations, these factors cause the enhancing attraction
between adjacent layers and forming irreversible aggregation or even restacking to graphite.

ip
t

It is also proposed that the interactions in tyrosine based GO, are intramolecular interactions
but in phenylalanine based GO, both intra and intermolecular interactions could existed, since
in GO+Phenylalanine pattern both increasing and decreasing in the layers distance were

cr

observed. Furthermore, in aromatic-aliphatic amino acids functionalized graphene, as a result

and becomes somewhat amorphous.

an

Fig. 5.

us

of - stacking and H-bonding interactions, the crystalline structure of graphene decreases

As shown in Fig. 6, the crystallographic orientations of the intercalation compounds

were found to vary significantly based on the structre of each aliphatic amino acid and their

properties. While GO exhibited the interplanar distance of 7.7 (2= 11.6), after
functionalization of GO with aliphatic amino acids, an increasing in d-spacing was monitored
from valine to methionine. These observations could be justified by amino acids structure,

functional groups and their chain length. The screening effect due to aliphatic amino acids,

te

that reduce the - stacking interaction between GO layers, plays an important role for the
exfoliation of GO. These enhancements are due to decreased interlayer interactions.

Ac
ce
p

Collectively, it is attained that, the aliphatic amino acids can be successfully intercalated into
GO and facilitating the formation of GO-amino acid intercalation compounds.
Fig. 6.

3.3.4. XPS spectroscopy

A more quantitative analysis has been done based on XPS spectra to investigate the

presence of functional groups on the amino acid functionalized GO. Fig. 7 displays the
survey XPS spectrum in the region of 0 to 1200 eV (a) and deconvoluted XPS spectra of the
C1s and O1s regions (b and c) of phenylalanine functionalized graphene. According to the
survey spectrum (Fig. 7a), three peaks centered at 284, 401 and 532 eV are observed which
can be assigned to C1s, N1s and O1s signals. In Fig. 7b, the C1s peak of functionalized
graphene located at 284 eV can be deconvoluted into four fitting curves with the binding
energy located at 284.1, 286.2, 287.5 and 289.03 eV which are assigned to C-C, C-N, amidic
9

Page 9 of 28

C=O and O-C=O, respectively [43,57,64,65]. Compared with GO XPS spectrum in

literatures, a high intensity peak at 286.7 eV is observed which is attributed to the epoxy
groups on GO surface [14,57,66], but here after functionalization of GO with phenylalanine,
a new peak at 286.2 eV is appeared which is due to nucleophilic interaction of phenylalanine

ip
t

NH2 groups and GO epoxy groups which causes to formation of C-N bands. Furthermore,
Fig. 7c presents the O1s peak of functionalized graphene with two fitting curves. There are
binding energies located at 531.1 and 532.4 eV which are assigned to O-C=O and C-OH,

cr

respectively. As it can be seen, the intensity of O-C=O groups is higher than that of C-OH
groups. This can be due to the inserted amino acid functionalities which contain O-C=O
of O-containing groups in phenylalanine.

an

Fig. 7.

us

groups. In another word, the higher intensity of O1s relative to C1s can be due to high degree

3.3.5. Dispersion stability in water

As a result of amino acid functionalization of graphene, dispersibility of the hybrids

was improved dramatically. Fig. 8 shows digital photographs of graphite, GO and

phenylalanine functionalized graphene which were dissolved in distilled water. Due to

graphite poor hydrogen bonding and van der Waals interaction, it does not disperse in water

te

and has tendency to aggregate or rope. In the case of amino acid functionalization of

Ac
ce
p

graphene, homogeneous and stable dispersion was observed which is more stable than GO.
Fig. 8.

3.3.6. Morphology studies

For observing the sheet structure and morphology of graphite, GO and its

functionalized form, microscopically and also to investigate the effect of functionalization on

the graphite surface by different amino acids, FE-SEM and TEM measurements were
performed. The results from FE-SEM images are in a good agreement with XRD analysis.
Fig. 9 displays the FE-SEM images of graphite and GO in different magnifications. As it can
be seen, the morphology and layer thickness of graphite sheets were completely changed after
oxidation. FE-SEM images of GO reveal a large increase in the thickness of graphene layers
throughout oxidization process. The observed outstanding increase in the thickness of
graphene sheets during the oxidization can be attributed to the formation of oxygen groups in
the basal plane of graphene.
10

Page 10 of 28

Fig. 9.
Fig. 10 demonstrates the FE-SEM micrographs of graphite and different types of
aliphatic amino acid functionalized graphene. The graphites image presents the sheet-like
structure with smooth surface. The pure graphite exhibits layered structures and becomes

ip
t

very thin. As it was reported in the XRD section, the graphene was more exfoliated and the dspacing was further increased in the presence of methionine, leucine and isoleucine. Their

cr

FE-SEM micrographs display flake-like structure with the large thickness, wrinkled edge and
crumble graphene sheet structure. The tiny particles on the surface of functionalized graphene

Fig. 10.

us

materials may be the proofs of amino acid existence on the graphene sheets.

an

Fig. 11 shows the FE-SEM images of aromatic-aliphatic amino acid based graphene.
As it can be seen, the FE-SEM micrographs of tyrosine based graphene certify the presence
of tubular structure that is as a result of scrolling effect of graphene layers. These images are

a complement to XRD data and show their nanotube-like structure. In Fig. 12 different
schematic kinds of graphene scrolling are shown.

te

Fig. 11.
Fig. 12.

Ac
ce
p

Fig. 13 shows the TEM micrographs of phenylalanine functionalized graphene. As

shown, we can distinctly find that it has a typical shape resembling the exfoliated crumpled
thin flake that its wrinkled structure is due to the presence of phenylalanine moieties between
layers after covalent functionalization.
Fig. 13.

Fig. 14 displays TEM images of tyrosine functionalized graphene. As it was observed

in XRD and FE-SEM sections, when GO was functionalized with tyrosine, the graphene
layers scrolled around each other which exhibit a unique structure between graphite and
multi-walled carbon nanotube. Accordingly, in TEM micrographs of tyrosine functionalized
graphene, it is obvious that the scrolled graphene was fabricated and it is also seen that the
graphene layers are more wrinkled than phenylalanine functionalized graphene. These images
revealed the presence of the tubular structure with different diameter ranging which the size
and shape of scrolled graphene are not uniform.
11

Page 11 of 28

Fig. 14.
3.3.7. Thermal stability
For further investigation of functionalized graphene properties, TGA analysis was
used as an effective instrument to determine the quantity of the grafted amino acid materials,

ip
t

because the forming covalent bonds between graphene sheet and amino acid moieties are
thermally stripped off in the temperature range of 150600 C. Fig. 15 shows TGA curves of

cr

graphite, GO, tyrosine functionalized graphene and methionine functionalized graphene

which indicate the thermal stability of surface functionalized graphene materials. As it can be

us

seen, pure graphite did not show any weight loss until 800 C. For GO, a slight weight loss
below 100 C is attributed to the removal of adsorbed water by O-containing functional
groups on GO sheets which is due to tightly bounded H2O molecules into GO stacked

an

structure. In addition, the weight loss around 200 C is attributed to decomposition of Ocontaining functional groups from the GO surface, yielding CO, CO2, and steam [3,67-69].

For comparison, one of the aliphatic functionalized graphene substances (methionine) and
one of the aromatic-aliphatic functionalized graphene materials (tyrosine) were choose. In
both cases, the weight loss of functionalized graphene is much smaller owing to decreased

amount of oxygen functional groups on their surfaces. For tyrosine functionalized graphene

te

and methionine functionalized graphene, the total weight loss is 23% and 54% respectively,
which is related to the pyrolysis of organic moieties on the graphene sheets, demonstrating a

Ac
ce
p

successful grafting of amino acid molecules to the graphene layers. As it observed, graphene
is more thermal stable when is functionalized with tyrosine amino acid than with methionine.
This phenomenon may be due to the existence of - stacking interactions between the
aromatic ring of tyrosine and graphene which cause to form more stacked structure.
Fig. 15.

4. Conclusions

An easy and effective approach was utilized for synthesis of different kinds of aliphatic and
aromatic-aliphatic amino acid functionalized graphene. The prepared materials were
characterized by FT-IR, Raman, XPS, XRD, FE-SEM, TEM and TGA analysis. Based on the
amino acid structure, functional groups and chain length, different changes on the structure
and morphology of graphene were observed. According to the XRD and FE-SEM results,
aliphatic amino acids cause an increase in interlayer spacing of graphene which the d-spacing
12

Page 12 of 28

is depend on their chain length. On the other hand, aliphatic-aromatic amino acid
functionalized graphene display distinct manner especially tyrosine based graphene. The
XRD patterns of tyrosine based graphene reveals a particular scrolled structure between
graphene and multi-walled carbon nanotubes that comes from rolling single layer of

ip
t

graphene. FE-SEM and TEM images also confirm this occurrence.

cr

Acknowledgements

us

We gratefully acknowledge the partial financial support from the Research Affairs Division
Isfahan University of Technology (IUT), Isfahan. Further partial financial support of Iran
Nanotechnology Initiative Council (INIC), National Elite Foundation (NEF) and Center of

an

Excellency in Sensors and Green Chemistry (IUT) is also gratefully acknowledged. Useful

help from Dr. H. farrokhpour is also gratefully acknowledged.

References

[1] B.-T. Zhang, X. Zheng, H.-F. Li, J.-M. Lin, Application of carbon-based nanomaterials in
sample preparation: A review, Anal. Chim. Acta, 784 (2013) 1-17.
Rev., 110 (2009) 132-145.

te

[2] M.J. Allen, V.C. Tung, R.B. Kaner, Honeycomb Carbon: A Review of Graphene, Chem.

Ac
ce
p

[3] T. Kuilla, S. Bhadra, D. Yao, N.H. Kim, S. Bose, J.H. Lee, Recent advances in graphene
based polymer composites, Prog. Polym. Sci., 35 (2010) 1350-1375.
[4] C.N.R. Rao, A.K. Sood, K.S. Subrahmanyam, A. Govindaraj, Graphene: The New TwoDimensional Nanomaterial, Angew. Chem. Int. Ed., 48 (2009) 77527777.
[5] S.K. Georgantzinos, G.I. Giannopoulos, N.K. Anifantis, Numerical investigation of
elastic mechanical properties of graphene structures, Mater. Des., 31 (2010) 46464654.
[6] R.F. Curl, S.H.W. Kroto, A. Geim, P. Wallace, GrapheneThe ultimate surface material,
Surf. Sci., 605 (2011) 1607-1610.
[7] D. Chen, H. Zhang, Y. Liu, J. Li, Graphene and its derivatives for the development of
solar cells, photoelectrochemical, and photocatalytic applications, Energy Environ Sci, 6
(2013) 1362-1387.

13

Page 13 of 28

[8] C. Xu, Y. Cao, R. Kumar, X. Wu, X. Wang, K. Scott, A polybenzimidazole/sulfonated

graphite oxide composite membrane for high temperature polymer electrolyte membrane fuel
cells, J. Mater. Chem., 21 (2011) 11359-11364.
[9] H. Chang, H. Wu, Graphene-Based Nanomaterials: Synthesis, Properties, and Optical and

ip
t

Optoelectronic Applications, Adv. Funct. Mater., 23 (2013) 1984-1997.

[10] O. Akhavan, E. Ghaderi, R. Rahighi, Toward Single-DNA Electrochemical Biosensing
by Graphene Nanowalls, ACS Nano, 6 (2012) 2904-2916.

cr

[11] O. Akhavan, E. Ghaderi, Flash photo stimulation of human neural stem cells on
graphene/TiO2 heterojunction for differentiation into neurons, Nanoscale, 5 (2013) 10316-

us

10326.

[12] J. Shen, N. Li, M. Shi, Y. Hu, M. Ye, Covalent synthesis of organophilic chemically

an

functionalized graphene sheets, J. Colloid Interface Sci., 348 (2010) 377383.

[13] T. Kuila, S. Bose, A.K. Mishra, P. Khanra, N.H. Kim, J.H. Lee, Chemical
functionalization of graphene and its applications, Prog. Mater Sci., 57 (2012) 1061-1105.

[14] Y.-K. Yang, C.-E. He, R.-G. Peng, A. Baji, X.-S. Du, Y.-L. Huang, X.-L. Xie, Y.-W.
Mai, Non-covalently modified graphene sheets by imidazolium ionic liquids for
multifunctional polymer nanocomposites, J. Mater. Chem., 22 (2012) 5666-5675.

[15] V. Singh, D. Joung, L. Zhai, S. Das, S.I. Khondaker, S. Seal, Graphene based materials:

te

Past, present and future, Prog. Mater Sci., 56 (2011) 11781271.

[16] F. Xia, D.B. Farmer, Y.-m. Lin, P. Avouris, Graphene Field-Effect Transistors with

Ac
ce
p

High On/Off Current Ratio and Large Transport Band Gap at Room Temperature, Nano Lett.,
10 (2010) 715-718.

[17] T. Szab, A. Szeri, I. Dkny, Composite graphitic nanolayers prepared by selfassembly between finely dispersed graphite oxide and a cationic polymer, Carbon, 43 (2005)
87-94.

[18] J.-Y. Wang, S.-Y. Yang, Y.-L. Huang, H.-W. Tien, W.-K. Chin, C.-C.M. Ma,
Preparation and properties of graphene oxide/polyimide composite films with low dielectric
constant and ultrahigh strength via in situpolymerization, J. Mater. Chem., 21 (2011) 1356913575.
[19] J.T. Robinson, M. Zalalutdinov, J.W. Baldwin, E.S. Snow, Z. Wei, P. Sheehan, B.H.
Houston, Wafer-scale Reduced Graphene Oxide Films for Nanomechanical Devices, Nano
Lett., 8 (2008) 3441-3445.
[20] G. Eda, M. Chhowalla, Graphene-based Composite Thin Films for Electronics, Nano
Lett., 9 (2009) 814-818.
14

Page 14 of 28

[21] X. Wang, L. Zhi, K. Mullen, Transparent, Conductive Graphene Electrodes for DyeSensitized Solar Cells, Nano Lett., 8 (2007) 323-327.
[22] W. Zhang, Q. Luo, X. Duan, Y. Zhou, C. Pei, Nitrated graphene oxide and its catalytic
activity in thermal decomposition of ammonium perchlorate, Mater. Res. Bull., 50 (2014) 73-

ip
t

78.
[23] C. Chen, W. Zhai, D. Lu, H. Zhang, W. Zheng, A facile method to prepare stable
noncovalent functionalized graphene solution by using thionine, Mater. Res. Bull., 46 (2011)

cr

583-587.

[24] M. Liu, Y. Duan, Y. Wang, Y. Zhao, Diazonium functionalization of graphene

us

nanosheets and impact response of aniline modified graphene/bismaleimide nanocomposites,

Mater. Des., 53 (2014) 466474.

an

[25] D.R. Dreyer, S. Park, C.W. Bielawski, R.S. Ruoff, The chemistry of graphene oxide,
Chem. Soc. Rev., 39 (2010) 228-240.

[26] B. Long, M. Manning, M. Burke, B.N. Szafranek, G. Visimberga, D. Thompson, J.C.

Greer, I.M. Povey, J. MacHale, G. Lejosne, D. Neumaier, A.J. Quinn, Non-Covalent

Functionalization of Graphene Using Self-Assembly of Alkane-Amines, Adv. Funct. Mater.,
22 (2012) 717-725.

[27] S.M. Kang, S. Park, D. Kim, S.Y. Park, R.S. Ruoff, H. Lee, Simultaneous Reduction and
Mater., 21 (2011) 108-112.

te

Surface Functionalization of Graphene Oxide by Mussel-Inspired Chemistry, Adv. Funct.

Ac
ce
p

[28] W.L. Zhang, Y.D. Liu, H.J. Choi, Graphene oxide nanocomposites and their
electrorheology, Mater. Res. Bull., 48 (2013) 4997-5002.
[29] D.K. Pandey, T. Fung, G. Prakash, R. Piner, Y.P. Chen, R. Reifenberger, Folding and
cracking of graphene oxide sheets upon deposition, Surf. Sci., 605 (2011) 1669-1675.
[30] Y. Wang, Z. Shi, J. Fang, H. Xu, J. Yin, Graphene oxide/polybenzimidazole composites
fabricated by a solvent-exchange method, Carbon, 49 (2011) 1199-1207.
[31] I.-Y. Jeon, D. Yu, S.-Y. Bae, H.-J. Choi, D.W. Chang, L. Dai, J.-B. Baek, Formation of
Large-Area Nitrogen-Doped Graphene Film Prepared from Simple Solution Casting of EdgeSelectively Functionalized Graphite and Its Electrocatalytic Activity, Chem. Mater., 23
(2011) 3987-3992.
[32] Y. Matsuo, T. Miyabe, T. Fukutsuka, Y. Sugie, Preparation and characterization of
alkylamine-intercalated graphite oxides, Carbon, 45 (2007) 10051012.

15

Page 15 of 28

[33] M.M. Gudarzi, F. Sharif, Enhancement of dispersion and bonding of graphene-polymer

through wet transfer of functionalized graphene oxide, eXPRESS Polym Lett, 6 (2012) 1017
1031.
[34] F. Zeng, Y. Kuang, Y. Wang, Z. Huang, C. Fu, H. Zhou, Facile Preparation of High-

ip
t

Quality Graphene Scrolls from Graphite Oxide by a Microexplosion Method, Adv. Mater., 23
(2011) 4929-4932.

[35] D. Roy, E. Angeles-Tactay, R.J.C. Brown, S.J. Spencer, T. Fry, T.A. Dunton, T. Young,

cr

M.J.T. Milton, Synthesis and Raman spectroscopic characterisation of carbon nanoscrolls,

Chem. Phys. Lett., 465 (2008) 254-257.
nanoribbons, Appl. Surf. Sci., 258 (2012) 1964-1970.

us

[36] D. Xia, Q. Xue, K. Yan, C. Lv, Diverse nanowires activated self-scrolling of graphene

an

[37] G. Wang, X. Shen, B. Wang, J. Yao, J. Park, Synthesis and characterisation of

hydrophilic and organophilic graphene nanosheets, Carbon, 47 (2009) 1359 1364.
[38] J. Liu, J. Tang, J.J. Gooding, Strategies for chemical modification of graphene and

applications of chemically modified graphene, J. Mater. Chem., 22 (2012) 12435-12452.

[39] J. Liu, L. Cui, D. Losic, Graphene and graphene oxide as new nanocarriers for drug
delivery applications, Acta Biomater., (2013).

[40] J.A. Nam, A.-A. Nahain, S.M. Kim, I. In, S.Y. Park, Successful stabilization of
9 (2013) 7996-8003.

te

functionalized hybrid graphene for high-performance antimicrobial activity, Acta Biomater.,

Ac
ce
p

[41] S. Gurunathan, J. Han, J.H. Kim, Humanin: A novel functional molecule for the green
synthesis of graphene, Colloids Surf. B. Biointerfaces, 111 (2013) 376-383.
[42]

A.M.

Pintoa,

I.C.

Goncalves,

F.D.

Magalhes,

Graphene-based

materials

biocompatibility: A review, Colloids Surf. B. Biointerfaces, 111 (2013) 188-202.

[43] Z. Mo, H. Gou, J. He, P. Yang, C. Feng, R. Guo, Controllable synthesis of functional
nanocomposites: Covalently functionalize graphene sheets with biocompatible L-lysine,
Appl. Surf. Sci., 258 (2012) 8623-8628.
[44] J. Guo, L. Ren, R. Wang, C. Zhang, Y. Yang, T. Liu, Water dispersible graphene
noncovalently functionalized with tryptophan and its poly(vinyl alcohol) nanocomposite,
Composites: Part B, 42 (2011) 2130-2135.
[45] C. Shan, H. Yang, D. Han, Q. Zhang, A. Ivaska, L. Niu, Water-Soluble Graphene
Covalently Functionalized by Biocompatible Poly-L-lysine, Langmuir, 25 (2009) 12030
12033.

16

Page 16 of 28

[46] O. Akhavan, E. Ghaderi, A. Esfandiar, Wrapping Bacteria by Graphene Nanosheets for

Isolation from Environment, Reactivation by Sonication, and Inactivation by Near-Infrared
Irradiation, J. Phys. Chem. B, 115 (2011) 6279-6288.
[47] M.J. Fernandez-Merino, L. Guardia, J.I. Paredes, S. Villar-Rodil, P. Solis-Fernandez, A.

ip
t

Martinez-Alonso, J.M.D. Tascon, Vitamin C Is an Ideal Substitute for Hydrazine in the

Reduction of Graphene Oxide Suspensions, J. Phys. Chem. C, 114 (2010) 6426-6432.

[48] Y. Wang, Z. Shi, J. Yin, Facile Synthesis of Soluble Graphene via a Green Reduction of

cr

Graphene Oxide in Tea Solution and Its Biocomposites, ACS Appl. Mater. Interfaces, 3
(2011) 1127-1133.

us

[49] O. Akhavan, M. Kalaee, Z.S. Alavi, S.M.A. Ghiasi, A. Esfandiar, Increasing the
antioxidant activity of green tea polyphenols in the presence of iron for the reduction of

an

graphene oxide, Carbon, 50 (2012) 3015-3025.

[50] O. Akhavan, E. Ghaderi, E. Abouei, S. Hatamie, E. Ghasemi, Accelerated differentiation

of neural stem cells into neurons on ginseng-reduced graphene oxide sheets, Carbon, 66

(2014) 395-406.

[51] W.S. Hummers, R.E. Offeman, Preparation of Graphitic Oxide, J. Am. Chem. Soc., 80
(1958) 1339-1339.

[52] K.N. Kudin, B. Ozbas, H.C. Schniepp, R.K. Prud'homme, I.A. Aksay, R. Car, Raman

te

Spectra of Graphite Oxide and Functionalized Graphene Sheets, Nano Lett., 8 (2007) 36-41.
[53] Y. Jin, S. Huang, M. Zhang, M. Jia, D. Hu, A green and efficient method to produce

Ac
ce
p

graphene for electrochemical capacitors from graphene oxide using sodium carbonate as a
reducing agent, Appl. Surf. Sci., 268 (2013) 541- 546.
[54] O. Akhavan, E. Ghaderi, Toxicity of Graphene and Graphene Oxide Nanowalls Against
Bacteria, ACS Nano, 4 (2010) 57315736.
[55] O. Akhavan, E. Ghaderi, S. Aghayee, Y. Fereydoonia, A. Talebi, The use of a glucosereduced graphene oxide suspension for photothermal cancer therapy, J. Mater. Chem., 22
(2012) 13773-13781.

[56] A.C. Ferrari, J.C. Meyer, V. Scardaci, C. Casiraghi, M. Lazzeri, F. Mauri, S. Piscanec,
D. Jiang, K.S. Novoselov, S. Roth, A.K. Geim, Raman Spectrum of Graphene and Graphene
Layers, Phys. Rev. Lett., 97 (2006) 187401-187404.
[57] H. Yang, F. Li, C. Shan, D. Han, Q. Zhang, L. Niu, A. Ivaska, Covalent
functionalization of chemically converted graphene sheets via silane and its reinforcement, J.
Mater. Chem., 19 (2009) 4632-4638.

17

Page 17 of 28

[58] A.C. Ferrari, Raman spectroscopy of graphene and graphite: Disorder, electronphonon
coupling, doping and nonadiabatic effects, Solid State Commun., 143 (2007) 47-57.
[59] I. Calizo, A.A. Balandin, W. Bao, F. Miao, C.N. Lau, Temperature Dependence of the
Raman Spectra of Graphene and Graphene Multilayers, Nano Lett., 7 (2007) 2645-2649.

ip
t

[60] K.S. Kim, Y. Zhao, H. Jang, S.Y. Lee, J.M. Kim, K.S. Kim, J.-H. Ahn, P. Kim, J.-Y.
Choi, B.H. Hong, Large-scale pattern growth of graphene films for stretchable transparent
electrodes, Nature, 457 (2009) 706-710.

cr

[61] N.I. Kovtyukhova, P.J. Ollivier, B.R. Martin, T.E. Mallouk, S.A. Chizhik, E.V.
Buzaneva, A.D. Gorchinskiy|, Layer-by-Layer Assembly of Ultrathin Composite Films from

us

Micron-Sized Graphite Oxide Sheets and Polycations, Chem. Mater., 11 (1999) 771-778.
[62] M.E. Achaby, F.Z. Arrakhiz, S. Vaudreuil, E.M. Essassi, A. Qaiss, Piezoelectric films, Appl. Surf. Sci., 258 (2012) 7668-7677.

an

polymorph formation and properties enhancement in graphene oxide PVDF nanocomposite

[63] C. Rajesh, C. Majumder, H. Mizuseki, Y. Kawazoe, A theoretical study on the

interaction of aromatic amino acids with graphene and single walled carbon nanotube, J
Chem. Phys., 130 (2009) 124911-124916.

[64] D.S. Everhart, C.N. Reilley, Chemical derivatization in electron spectroscopy for

te

Anal. Chem., 53 (1981) 665-676.

chemical analysis of surface functional groups introduced on low-density polyethylene film,

[65] O.C. Compton, B. Jain, D.A. Dikin, A. Abouimrane, K. Amine, S.T. Nguyen,

Ac
ce
p

Chemically Active Reduced Graphene Oxide with Tunable C/O Ratios, ACS Nano, 5 (2011)
43804391.

[66] S. Sheshmani, M.A. Fashapoyeh, Suitable Chemical Methods for Preparation of

Graphene Oxide, Graphene and Surface Functionalized Graphene Nanosheets, Acta Chim.
Slov., 60 (2013) 813-825.

[67] J.-Y. Kong, M.-C. Choi, G.Y. Kim, J.J. Park, M. Selvaraj, M. Han, C.-S. Ha, Preparation
and properties of polyimide/graphene oxide nanocomposite films with Mg ion crosslinker,
Eur. Polym. J., 48 (2012) 1394-1405.
[68] O.C. Compton, D.A. Dikin, K.W. Putz, L.C. Brinson, S.T. Nguyen, Electrically
Conductive Alkylated Graphene Paper via Chemical Reduction of Amine-Functionalized
Graphene Oxide Paper, Adv. Mater., 22 (2010) 892896.
[69] Y. Yang, T. Liu, Fabrication and characterization of graphene oxide / zinc oxide
nanorods hybrid, Appl. Surf. Sci., 257 (2011) 8950-8954.

18

Page 18 of 28

Figure captions
Fig. 1. Synthesis of GO and functionalized graphene with different types of amino acids.
Fig. 2. FT-IR spectra of the pure graphite, GO and different amino acid functionalized
graphene and FT-IR comparing of phenylalanine and tyrosine functionalized graphene with

ip
t

their attributed amino acids.

Fig. 3. Raman spectra of a: GO and b: phenylalanine functionalized graphene.

cr

Fig. 4. XRD patterns of graphene and GO.

us

Fig. 5. XRD patterns of GO and different aromatic-aliphatic amino acid based graphene.
Fig. 6. XRD patterns of GO and different aliphatic amino acid based graphene.

an

Fig. 7. a: The survey XPS spectrum and deconvoluted XPS spectra of the C1s and O1s
regions (b and c) of phenylalanine functionalized graphene.

Fig. 8. Digital photograph of graphite, GO and phenylalanine functionalized graphene. All

the samples were dispersed in distilled water.

Fig. 9. FE-SEM images of graphite and GO.

functionalized graphene.

te

Fig. 10. FE-SEM micrographs of pure graphite and different aliphatic amino acid

Ac
ce
p

Fig. 11. FE-SEM micrographs of different aromatic-aliphatic amino acid functionalized

graphene.

Fig. 12. Graphene scrolling types.

Fig. 13. TEM micrographs of phenylalanine functionalized graphene.

Fig.14. TEM micrographs of tyrosine functionalized graphene.
Fig. 15. TGA curves of graphite, GO, tyrosine functionalized graphene and methionine
functionalized graphene.

19

Page 19 of 28

ip
t
cr
us
an
M
Ac
ce
p

te

Fig. 1

Fig. 2
20

Page 20 of 28

ip
t
us

cr
Ac
ce
p

te

an

Fig. 3

Fig. 4

Fig. 5
21

Page 21 of 28

ip
t
cr
us
an

Ac
ce
p

te

Fig. 6

Fig. 7
22

Page 22 of 28

ip
t
cr
us
an
M
Ac
ce
p

te

Fig. 8

Fig. 9

23

Page 23 of 28

ip
t
us

cr
Ac
ce
p

te

an

Fig. 10

Fig. 11

24

Page 24 of 28

cr

ip
t
Ac
ce
p

te

an

us

Fig. 12

Fig. 13

25

Page 25 of 28

ip
t
us

cr
Ac
ce
p

te

an

Fig. 14

Fig. 15

26

Page 26 of 28

27

Page 27 of 28

te

Ac
ce
p
us

an

cr

ip
t

Highlights
Bio-functionalized graphene materials were synthesized through a simple technique.
Biocompatible aliphatic and aromatic amino acids were used for graphene

functionalization.
Based on amino acid structure different changes on graphene structure and

ip
t

morphology were observed.

Ac
ce
p

te

an

us

cr

Graphene scrolling was observed by tyrosine functionalized graphene.

28

Page 28 of 28