J Nanopart Res (2015) 17:88
DOI 10.1007/s11051-015-2888-6
RESEARCH PAPER
Nano-graphene oxide carboxylation for efficient
bioconjugation applications: a quantitative optimization
approach
Rana Imani • Shahriar Hojjati Emami •
Shahab Faghihi
Received: 17 September 2014 / Accepted: 27 January 2015
Ó Springer Science+Business Media Dordrecht 2015
Abstract A method for carboxylation of graphene
oxide (GO) with chloroacetic acid that precisely
optimizes and controls the efficacy of the process
for bioconjugation applications is proposed. Quan-
tification of COOH groups on nano-graphene oxide
sheets (NGOS) is performed by novel colorimetric
methylene blue (MB) assay. The GO is synthesized
and carboxylated by chloroacetic acid treatment
under strong basic condition. The size and mor-
phology of the as-prepared NGOS are characterized
by scanning electron microscopy, transmission
electron microscopy (TEM), and atomic force
microscopy (AFM). The effect of acid to base
R. Imani  S. H. Emami (&)
Department of Biomedical Engineering, Amirkabir
University of Technology, 15875/4413 Tehran, Iran
e-mail: semami@aut.ac.ir
R. Imani  S. Faghihi (&)
Tissue Engineering and Biomaterials Division, National
Institute of Genetic Engineering and Biotechnology,
14965/161 Tehran, Iran
e-mail: shahabeddin.faghihi@mail.mcgill.ca;
sfaghihi@nigeb.ac.ir
molar ratio on the physical, chemical, and morpho-
logical properties of NGOS is analyzed by Fourier-
transformed infrared spectrometry (FTIR), UV–Vis
spectroscopy, X-ray diffraction (XRD), AFM, and
zeta potential. For evaluation of bioconjugation
efficacy, the synthesized nano-carriers with different
carboxylation ratios are functionalized by oc-
taarginine peptide sequence (R8) as a biomolecule
model containing amine groups. The quantification
of attached R8 peptides to graphene nano-sheets’
surface is performed with a colorimetric-based
assay which includes the application of 2,4,6-
Trinitrobenzene sulfonic acid (TNBS). The results
show that the thickness and lateral size of nano-
sheets are dramatically decreased to 0.8 nm and
50–100 nm after carboxylation process, respective-
ly. X-ray analysis shows the nano-sheets interlaying
space is affected by the alteration of chloroacetic
acid to base ratio. The MB assay reveals that the
COOH groups on the surface of NGOS are
maximized at the acid to base ratio of 2 which is
confirmed by FTIR, XRD, and zeta potential. The
TNBS assay also shows that bioconjugation of the
optimized carboxylated NGOS sample with oc-
taarginine peptide is 2.5 times more efficient
compared to bare NGOS. The present work
provides evidence that treatment of GO by
chloroacetic acid under an optimized condition
would create a functionalized high surface area
nano-substrate which can be used for subsequent
efficient bioconjugation applications.
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Graphical Abstract
Keywords Graphene oxide  Carboxylation 
Methylene blue  Bioconjugation  Nanobiomaterials
Introduction
The employment of nano-biomaterials for fabrication
of biomedical devices has generated remarkable
achievements in recent years (Das et al. 2014). The
application of carbon-based materials has opened new
opportunities for producing novel functional nano-
materials (Cha et al. 2013). Due to the unique
electrical, thermal, optical, mechanical, and biological
properties, graphene, a fascinating 2D material has
attracted tremendous attention (Chung et al. 2013). As
a biocompatible material, graphene and its derivatives
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J Nanopart Res (2015) 17:88
have found many applications in biomedical research-
es such as drug delivery, cellular imaging, photother-
mal therapy, gene therapy, cancer targeting,
biosensors, and tissue engineering(Akbari et al.
2014; Goenka et al. 2014; Mao et al. 2013; Najafabadi
et al. 2014; Pinto et al. 2013; Tang et al. 2013; Yang
et al. 2013b). The major advantage of graphene-based
nano-materials is their high specific surface area
which enables a high-density bio-functionalization
and biomolecules loading (Yang et al. 2013a). Various
graphene-based nano-materials have been used as
substrate to interact with nucleic acids (DNA or RNA),
peptides, proteins, and cells (Shen et al. 2012, 2010;
Wang et al. 2011b; Zhang et al. 2012). The function-
alization of graphene sheets is therefore of crucial
importance in their application and to control their
biocompatibility and cytotoxicity.
J Nanopart Res (2015) 17:88
Scheme 1 An illustration of common routes for carboxyl-
mediated GO bio-conjugations. Covalently conjugation of
a Poly ethylene glycol (branched or linear) for increased water
solubility and bio-distribution, b amine-contained natural or
synthetic polymers e.g. Polyethylene imine, chitosan, c targeting
agent like folic acid for cancer therapy, d antibodies for
biosensor designing and cell targeting, e fluorescence agent for
The capacity of biomolecules loading and immo-
bilization of graphene for examples as a drug carrier,
gene vehicle, or biosensor platform is regulated by the
amounts and types of its surface functional groups
(Yang et al. 2013a). The oxidation of graphene to
graphene oxide (GO) is the simplest way to introduce
functional groups onto the graphene surface. This
would increase the hydrophilicity of the graphene and
the distance between sheets, therefore, facilitates the
exfoliation and dispersion of GO sheets (Sun 2012).
The GO sheets have chemically reactive oxygen
functionalities mainly including carboxylic acid,
epoxy and hydroxyl groups (Dreyer et al. 2010). The
carboxylic acid groups have been utilized as a reactant
site for immobilization or conjugation of peptides and
proteins (Jin et al. 2012; Shen et al. 2010), enzymes (Li
et al. 2013), antibodies (Zhang et al. 2014), targeting
agents (Huang et al. 2010), and amine containing
natural or synthetic polymers (Bao et al. 2011; Kim
et al. 2011) (Scheme 1). The conjugation of positively
Page 3 of 15 88
bioimaging, f amineated nano-particles e.g., Fe2O3 for cellular
imaging, g proteins and peptide e.g. enzymes, electrostatically
conjugation of h cationic natural or synthetic polymers,
i positively charged nano-particles, j cationic linker molecules
for loading negatively charged biomolecules e.g. nucleic acids
DNAs, RNAs
charge molecules is mediated through negatively
charged carboxyl groups while the covalent conjuga-
tion often requires activation of the acidic groups. The
subsequent addition of nucleophilic species, such as
amines or hydroxyls to activated carboxyl groups,
produces covalently attached molecules to GO plate-
lets and forms amides or esters. A deficiency in the
amount of carboxyl groups on the basal plane of GO
will limit biomolecules immobilization to the edge of
graphene sheets (Yuge et al. 2008). The conversion of
other oxygen containing functional groups like hy-
droxyl or ether on the basal plane of GO nano-sheets
would provide more active sites for further
functionalization.
Chloroacetic acid has been widely used to trans-
form unreactive hydroxyls into carboxylate groups for
subsequent conjugations. It is reported that enrichment
of surface carboxyl groups of GO by chloroacetic acid
treatment maximizes the interaction between the GO
and Ag nano-particles for photocatalytic application
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88 Page 4 of 15
(Zhang et al. 2013). Liu et al. have increased the basal
oxygenated groups of GO by hydrobromic acid/oxalic
acid in order to create constructed GO–polyaniline
composites through in situ polymerization (Liu et al.
2012). Yue et al. have employed carboxylation
process to enhance conjugation of fluorescent mole-
cules on the GO which can be used as a probe for
tumor diagnosis (Yue et al. 2013). The carboxylation
process was also utilized in other many studies for
PEGylation of GO as a drug delivery vehicle (Dong
et al. 2010; Liu et al. 2008; Sun et al. 2008; Wen et al.
2012). Conjugation of cell penetrating peptides along
with caspase-3 peptide on the carboxylated GO sheets
was also introduced by Wang et al. as an intracellular
protease sensor (Wang et al. 2011a).
Besides from functionalization, controlling car-
boxylation process and quantification of carboxyl
groups on the surface of GO nano-sheets could serve
as a useful tool to direct subsequent bioconjugations
since further bioconjugation directly depends on the
available conjugation sites. In the other word, before
any conjugation of biomolecule to the graphene oxide
nano-sheets, estimation of surface density of func-
tional group which is responsible for covalent attach-
ment provide good insight to design subsequent
reaction condition. While much research has been
devoted to the functionalization and subsequent bio-
conjugation processes (Kim et al. 2011; Yue et al.
2013), there has been no investigation on quantitative
optimization of carboxylation process on the surface
of GO nano-sheets. However, the functional groups
distribution on graphene oxide has been examined by
numerous methods such as solid-state NMR (Gao et al.
2009), FTIR (Bagri et al. 2010), NEXAFS (Lee et al.
2012), and XPS (Ganguly et al. 2011); some of these
techniques could quantify carboxyl groups rather than
other surface oxygenated groups. In this study, the
NGOS are synthesized by modified Hummer method
and the resultant product is oxidized by chloroacetic
acid under basic condition. The effects of chloroacetic
acid/NaOH ratios on the carboxylation process of GO
nano-sheets are also investigated. The process is
characterized using Fourier transform infrared spec-
troscopy (FTIR), UV–Vis spectroscopy, zeta potential
analysis, atomic force microscopy (AFM), scanning
electron microscopy (SEM), transmission electron
microscopy (TEM), and X-ray diffraction (XRD).
Finally, the concentration of carboxyl group per
weight of NGOS is quantitatively measured by MB
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J Nanopart Res (2015) 17:88
calorimetric assay as a simple and efficient technique.
For evaluation of bioconjugation efficacy of carboxy-
lated NGOS, octaarginine peptide was selected as a
biomolecule model containing amine groups. The
synthesized nano-carriers with different carboxylation
ratios were functionalized by octaarginine peptide
sequence (R8). The attachment of R8 peptides to
graphene nano-sheets’ surface is quantified with a
colorimetric-based assay which includes the applica-
tion of 2,4,6-Trinitrobenzene sulfonic acid (TNBS).
Materials and methods
Materials
All chemical materials involved in GO synthesis were
purchased from Sigma-Aldrich (Japan). Chloroacetic
acid, Methylene blue (MB), 1-ethyl-3-[3-dimethy-
laminopropyl] carbodiimide hydrochloride (EDAC),
N-hydroxysulfosuccinimide (NHS) and 4-morpholi-
noethanesulfonic acid (MES) were obtained from
Merck (Germany). Octaarginine was obtained from
GL Biochem LTD (Shanghai). Sodium bicarbonate,
sodiumdodecyl sulfate (SDS), and 2,4,6-Trinitroben-
zene sulfonic acid (TNBS) used for TNBS assay were
purchased from Merck (Germany).
Preparation of NGOS
The NGOS was prepared based on a modified
Hummer method (Hummers and Offeman 1958). In
a typical experiment, natural graphite powder (1 g)
was suspended in 120 ml of sulfuric acid (98 %). The
mixture was cooled in an ice bath followed by addition
of NaNO3 (500 mg) under moderate stirring
(200 rpm). After 60 min, KMnO4 (6 g) was added
and the mixture was heated to reach 35 °C under
constant stirring condition. After 48 h, the brownish
green solution became too viscose to stir. Double
distilled water (DDW) was slowly added to the
reaction to keep the temperature at 70 °C for 1 h.
Finally, 30 % H2O2 (10 ml) was added to the mixture
until the color was changed to bright yellow. The
mixture was rested for 2 days to precipitate GO nano-
sheets. The supernatant was removed and precipitated
powder was washed ten times with 0.5 M aqueous
HCl to remove metal ions followed by washing with
DDW to remove the acid residues. To achieve nano-
J Nanopart Res (2015) 17:88
sized mono layer GO sheet, the suspension was
dispersed by probe-typed ultrasonic treatment (Mi-
crosonTM, XL2000, USA, 200 W, 2 h). The resultant
brown solution was freeze dried (Christ-Alpha 1–4,
Biotech International, Germany) to obtain a fine nano-
graphene oxide powder.
Carboxylation of nano-graphene oxide sheets
(NGOS-COOH)
Carboxylic acid-functionalized nano-graphene oxide
sheets (NGOS-COOH) were obtained through reac-
tion with chloroacetic acid under strong basic condi-
tions to transform the hydroxyl, epoxide, and ester
groups into carboxylic acid (COOH) moieties. In order
to optimize the oxidation process, four different
concentrations of chloroacetic acid (0.5, 1, 2, and
3 M) were used. The prepared samples were called as
OXG0.5, OXG1, OXG2, and OXG3, respectively.
For carboxylation, 1 mg of NGOS was dispersed in
deionized water (1 ml) under probe-type sonication
(100 W, 30 min) to obtain a clear solution. Chloroace-
tic acid solutions were prepared in 4 M NaOH and the
NGOS aqueous suspension was immediately added to
these solutions. The final concentration of NGOS in
chloroacetic acid solution was 0.2 mg/ml. The suspen-
sion was allowed to react for 75 min under bath-
sonication (Fisher Scientific Co., USA) at room tem-
perature in order to transform –OH groups to –COOH
via conjugation of acetic acid moieties. The NGOS
–COOH solution was centrifuged and washed five times
with DDW. The suspension was transferred to a dialysis
bag (12 kDa, Sigma-Aldrich, Japan) and dialyzed
against DDW to remove the excess reactant and obtain
a well-dispersed NGOS-COOH aqueous solution.
NGOS and NGOS –COOHs characterizations
The samples of NGOS and NGOS-COOHs were pressed
into the potassium bromide pellets and examined with
Fourier transform infrared (FTIR) spectrophotometer
within the range of 3,600–500 cm-1 at a resolution of
4 cm-1 (German Nicolet FTIR Nexus-670 IR). UV–Vis
absorption spectra of both samples were also obtained
using a Neosys-2000, Scinco Co. Ltd., Korea spec-
trophotometer at room temperature (using quartz cuvette
with a 1-mm gap; 0.5 ml of 0.1 mg/ml sample solution).
The morphology of NGOS and NGOS-COOH deposited
from a dilute aqueous dispersion (0.01 mg/ml) on a
Page 5 of 15 88
freshly cleaved mica was determined by an atomic force
microscope (AFM, Nanoscope III Multimode,VEECO)
in tapping mode and scanning electron microscopy
(SEM, AIS2300C, Soren technology, Korea) operated at
220 kV as well as transmission electron microscope
(TEM, Philips169 CM200 200 kV). The X-ray diffrac-
tion (XRD) analysis of the GO-deposited surface was
performed using a diffractometer (EQUINOX300, Inel,
France) at 2h ranging from 5° to 40° using CuKa
radiation (40 kV, 100 mA). Finally, zeta potential of
NGOS and NGOS-COOHs samples (0.01 mg/ml, pH
6.1) was measured using a Zetasizer Nano-ZS-90
(Malvern Instruments, UK).
MB assay
Quantification of carboxyl groups of GO and carboxy-
lated samples was carried out by MB colorimetric assay.
The bleaching of MB diluted solution correlates the
chemical reaction between the MB molecules and
COOH groups in the solution (Nguyen 2012). For
preparing MB standard curve, UV–Vis absorption
spectra of MB solution at various concentrations
(0.27–1.6 lg/ml) were recorded. The peak at 664 nm
was selected for plotting the calibration curve of the MB
solution. Subsequently, GO samples with defined weight
were immersed into the MB solution (pH = 8.5) for
10 min. The GO samples were then removed from the
colorless MB solution by centrifugation (14,000 rpm,
15 min). By adding same amount of DDW (pH = 8.5)
to the pellet and re-suspending, the centrifugation step
was repeated 3 times to removal all non-bonded dyes.
The absorbance of the supernatant was read at 664 nm to
estimate the concentration of unreacted MBs using the
standard curve. Since each MB molecule can only
interact with one COOH group, the difference between
the MB concentration before and after the reaction was
used to measure the COOH concentrations. Finally the
number of MB molecules which is equal to the COOH
functional groups in the samples was calculated.
Biomolecules conjugation
For evaluation of bioconjugation efficacy of carboxy-
lated NGOS, octaarginine peptide was selected as a
biomolecule model. The synthesized nano-carriers
with different carboxylation ratios were functional-
ized by octaarginine peptide sequence (R8) through
two steps diimide-activated amidation under ambient
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conditions (Shen et al. 2010). In the first step, 10 mg of
NGOS-COOH was suspended in 10 ml of deionized
water by sonication of the mixture for 1 h. Subse-
quently, 5 ml of a 500-mM MES buffer solution
(pH * 6.1) and 5 ml of a 50-mg/ml NHS aqueous
solution were added to the above mentioned suspen-
sion. After 15 min of fast stirring, 6-ml fresh EDAC
aqueous solution (10 mg/l was added to the mixture at
room temperature. After 30 min stirring, the suspen-
sion was centrifuged and rinsed thoroughly with a
50-mM MES buffer solution (pH * 6.1) to remove
excess reactants. In the second step, the estered nano-
sheets were re-dispersed in 5 ml of a 50 mM MES
buffer solution (pH * 6.1) and 15 lmol of R8 was
added to the mixture. After shaking the mixture on a
platform shaker (150 rpm) at room temperature for
24 h, the suspension was centrifuged and washed with
50-mM MES buffer solution (pH * 6.1) to remove
non-bonded peptides. The peptide functionalized
nano-sheets (R8-NGOSs) were dispersed in 5-ml
deionized water for further measurements.
TNBS assay
For quantification of attached R8 peptides to
graphene nano-sheets’ surface, a colorimetric-based
assay was used which employed 2,4,6-Trinitroben-
zene sulfonic acid (TNBS, Thermo Scientific Pierce).
The R8 peptide solutions were first used to produce a
standard curve for quantification of R8 binding onto
the graphene nano-sheets. In brief, 0.5-ml TNBS in
0.1-M sodium bicarbonate (pH 8.5, 0.01 %, w/v) was
added to 1 ml of R8 solutions having different
concentrations (10 to 100 lg/ml). The mixtures were
then incubated at 37 °C for 2 h followed by the
addition of 0.5 ml of 10 % sodiumdodecyl sulfate
(SDS) and 0.25 ml of 1-M HCl to each solution. The
absorbance was read at 335 nm and plotted against
R8 concentration for each sample. After the addition
of peptide-conjugated samples (0.5 mg/ml) to TNBS
solution, the peptide concentration per mg of GO
nano-sheets was calculated for each sample.
Statistical analysis
Data were first analyzed by analysis of variance
(ANOVA); when statistical differences were detected,
Tukey’s Multiple Comparison test was performed.
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J Nanopart Res (2015) 17:88
Data are reported as mean ± SD at a significance level
of p \ 0.05.
Results and discussion
FTIR spectroscopy
The chemical structures of synthesized NGOS and its
COOH-functionalized derivatives were characterized
by FTIR spectroscopy (Fig. 1). The comparison
between graphite and NGOS spectra confirms suc-
cessful oxidation reaction of graphene. The charac-
teristic peaks at 3,410, 1,733, 1,620, and 1,420 cm-1
correspond to vibration mode of OH, C=O, COOH,
and aromatic C=C functional groups in NGOS,
respectively (Dreyer et al. 2010). After chloroacetic
acid activation, the resulting NGOS-COOH deriva-
tives showed stronger absorption band at 1,620 cm-1
which indicates formation of additional carboxylate
moieties. The peak at *1,150 cm-1 is attributed to the
vibration mode of epoxide groups (C–O–C) which is
more pronounced in the NGOS spectra compared to
carboxylated samples where the peak is almost
vanished. This is possibly duo to the synergistic
effects of chloroacetic acid on both epoxide and
hydroxyl groups (Zhang et al. 2011).
The oxidation of GO by chloroacetic acid is
expected to convert –OH and surface ether groups
(C–O–C) into carboxyl ones (–COOH). By interaction
of surface –OH groups with chloroacetic acid, C–O–
COOH would be replaced with C–OH. The C–O–C
epoxide ring on the basal plane of GO would also be
firstly opened under basic condition and converted
to –OH and subsequently replaced with C–O–COOH.
As a result, it is expected to observe a decrease in free
–OH (hydroxyl) stretch around 3,200–3,500 cm-1 and
an increase related to the –OH stretch around
2800-3200 cm-1. As depicted in Fig. 1 by increasing
oxidation ratio to OXG2, the related peak of –OH was
shifted from 3,200–3,500 cm-1 to 3,100–3,200 cm-1.
Additionally, after chloroacetic acid reaction with
NGOS, two peaks at 2,854 and 2,925 cm-1 were
appeared in the IR spectrum. The intensity of these
peaks was enhanced by increasing chloroacetic acid
concentration up to 2 M (OXG2). These two bands are
correlated to the symmetric and asymmetric stretching
modes of -CH2- groups in chloroacetic acid. Further-
more, the peak at 1,390–1,400 cm-1 is attributed to
J Nanopart Res (2015) 17:88
Fig. 1 FTIR spectra of
NGOS and carboxylated
samples. The inset presents
the FTIR spectrum of
graphite
deformation vibration of -CH2- group (Szabó et al.
2006b). The small peak around 2,360 cm-1 could be
associated with the O–H stretch from strongly hydro-
gen-bonded –COOH groups (Davis et al. 1999). These
are the confirmation for chemically attachment of
many –O–CH2–COOH to the NGOS sheets.
Morphological characterization of NGOS
To characterize size and morphology of NGOS- and
carboxyl-functionalized samples having different de-
grees of carboxylation, AFM, SEM, and TEM were
employed (Fig. 2a–c). The results indicated that pre-
pared GO have sheet-like morphology with the size of
250–400 nm. The TEM analysis provided more detailed
morphological insight on the as-prepared GO sample
(Fig. 2c). Based on the TEM image, typical wrinkle
morphology for the NGOS was observed. The thickness
of unmodified NGOS was measured (at least for 10
separate sheets) based on the height profile of the AFM
images was 1–1.5 nm which is corresponded to 1–2
Page 7 of 15 88
layer of graphene sheets (Fig. 2a). The carboxylated
samples’ mean thickness was around 0.8 nm indicating
the formation of single-layered sheets (Fig. 2a). Due to
the strong basic condition in carboxylation process, the
modified samples showed reduced size of 50–100 nm
(Szabó et al. 2006b). However, the increase in the
chloroacetic acid concentration and degree of oxidation
process had little effect on the size and thickness of the
NGOS-COOH. The decrease that was detected in size of
the carboxyl-functionalized samples was correlated to
the sonication process involved in the activation step.
The reduced size and thickness observed in carboxylat-
ed GO samples could be more attractive for biological
interactions particularly gene and drug deliveries since
the larger surface area for molecular conjugation would
be obtained (Chang et al. 2011; Mu et al. 2012).
UV–Vis spectroscopy analysis
The carboxylation of NGOS was further characterized
by UV–Vis spectroscopy. As shown in Fig. 2c, there is
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Fig. 2 Size and morphological characterization of synthesized
NGOS and NGOS-COOH, a AFM image of NGOS (up) and
carboxylated GO (down, OXG1). b SEM image of as-prepared
a characteristic peak at 231 nm which is corresponded
to p-p* transitions of C=C bonds, and a shoulder at
300 nm corresponded to n-p* transitions of carbonyl
groups (Yang et al. 2011). The carboxylated samples
showed a much higher absorbance in the Vis–NIR
range compared to the NGOS. More importantly, the
absorption peak at 300 nm disappeared. The sig-
nificant increase in absorbance led to a darkening of
the solution as it is clearly seen in Fig. 2c. The color
change could be attributed to the restoration of the
electronic conjugation within the graphene sheets as it
was reported in the other study (Sun et al. 2008). Here,
the restoration is possibly related to the opening of the
epoxide rings and hydrolysis of esters on the GO under
the basic conditions during the activation process. This
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J Nanopart Res (2015) 17:88
NGOS, c TEM image of NGOS, d Uv–vis spectra of NGOS and
carboxylated NGOS in aqueous solution
could result to some local changes in the microstruc-
ture of NGOS as well as released local. This would in
turn increase the optical density absorption which was
detected in the Vis–NIR range.
XRD analysis
The layer distances, the extent of sheet folding, and
structure disruptions of graphite are highly different
from GO and carboxylated samples (Zhang et al.
2009). Therefore, all the samples powders were
characterized by XRD for more structural analysis
and to investigate the degree of oxidation and
carboxylation GO. The XRD patterns are shown in
Fig. 3 that confirm the chemical oxidation of the
J Nanopart Res (2015) 17:88
Fig. 3 XRD patterns of
graphite, GO, and
carboxylated samples
exfoliated graphite and the formation of GO sheets. The
XRD patterns also revealed the typical diffraction peak
of native graphite at 2h = 26.8. The oxidation process
is resulted the formation of hydroxyl and epoxy as well
as carboxyl groups between the graphite sheets which
are mainly located on the center and lateral sides of the
sheets, respectively. The appearance of these functional
groups weakens the Van der Waals forces between the
graphene sheets in the exfoliated GO (Karthika et al.
2012). Therefore, the diffraction peak of NGOS at
2h = 12.3 with no peak at 26.8 from graphite lattice
indicates that the exfoliation process under strong basic
condition has increased the sheets spacing. The
carboxylated samples showed much weaker and broad-
er diffraction peak (2h = 12.3) than NGOS indicating
increased interlayer spacing mostly due to the inclusion
of additional functional groups as well as the basic
Page 9 of 15 88
condition. As it can be seen, the intensity of the peak
decreased gradually as the degree of oxidation in-
creased (OXG0.5–OXG2) (Fig. 3). This would suggest
more intercalation of chloroacetic acid molecules
which resulted in a much higher expansion of the
interlayer spacing and corrugated structure of the GO
sheets. It is known that the extent of sheets folding
increases with the degree of carboxylation (Szabó et al.
2006a; Zhang et al. 2009). The XRD results are in
agreement with the FTIR data which reflect less
carboxylation progress for the OXG3 sample.
Zeta potential and dispersion stability analyses
The surface charges of the NGOS before and after
carboxylation process with chloroacetic acid were
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Fig. 4 Zeta potential
(0.01 mg/ml, pH 6.1) and
colloidal stability of
a NGOS and b NGOS-
COOH (OXG2) after 30 day
of incubation at room
temperature
determined by Zeta Sizer. After the treatment of the
NGOS by 2 M chloroacetic acid, a noticeable decrease
of the zeta potential to -46 mv was detected (Fig. 4).
This is mostly due to the introduction of more
carboxylic acid groups onto the surface of the NGOS.
As for the stability of the samples, it was revealed that
all carboxylated samples were remained completely
dispersed for 3 months without any precipitation.
However, unmodified NGOS showed colloidal insta-
bility after this period of time (Fig. 4, inset). This
could be due to the hydrophilic nature of the
carboxylated graphene sheets which results in exfo-
liation of the dried powder in water and formation of a
stable colloidal suspension under a mild sonication
condition. Zeta potential is an important factor for
characterization of dispersion stability of colloidal
systems as it could directly influence the electrostatic
interaction between the graphene sheets (Chowdhury
et al. 2013). Furthermore, increasing the carboxyl
groups of the surface that induces high negative
charges would allow the binding of more positively
charged polymers, peptides, proteins, and nano-parti-
cles to GO sheets via electrostatic interactions.
Methylene blue assay
The methylene blue (MB) assay is a common method
in wood industry to measure carboxyl groups of wood
pulps or cellulose derivatives (Khazraie Shoulaifar
et al. 2012; Šauperl et al. 2003). Here, MB assay was
utilized to quantify COOH functional groups of the
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J Nanopart Res (2015) 17:88
NGOS and carboxylated derivatives. The bleaching of
MB diluted solution is based on the chemical reaction
between the MB molecule and a COOH group in the
solution (Nguyen 2012) as represented in Fig. 5. The
chemical reaction occurs between one MB molecule
and one carboxyl group. It is therefore possible to
estimate the number of carboxyl groups per weight
unit of the GO samples accurately. The bleaching
reaction in acidic media will form a leucomethylene
molecule which is colorless as it is presented in Fig. 5.
The resultant solutions from the bleaching of MB
with carboxyl groups of graphene oxide sheets were
characterized by UV–Vis spectroscopy (Fig. 6). The
UV–Vis absorption spectrum of MB solution showed
two peaks at 612 and 664 nm (Fig. 6a). The second
peak (664 nm) was selected in order to plot a
calibration curve for the MB solution (Fig. 6a, inset).
Figure 6 depicts that the absorbent peak intensities
increased as the concentration of MB solution in-
creased from 0.27 to 1.6 lg/ml. A linear relation
between the MB concentration and the absorption
values was detected in this concentration range
(Fig. 6a). The interaction between MB molecules
and COOH groups in the NGOS was reduced the
absorption values as indicated in Fig. 6b. The differ-
ence in concentration of the MB solution before and
after the reaction with NGOS could be easily
converted to the number of MB molecules which is
equal to the number of COOH groups. Based on the
calibration curve, the molarity of COOH groups in
each mg of samples could be calculated according to
the Eq. 1:
J Nanopart Res (2015) 17:88
Fig. 5 The representation
of MB assay consist of
bleaching of MB solution as
well as the reaction of MB
molecule and carboxyl
groups in GO
ð0:3219  Y1  Y2 þ 0:0903Þ
MCOOH ¼ ; ð1Þ
ð107:3  Ms Þ
where MCOOH is the molarity of COOH group
(lM/mg), Y1 and Y2 are the MB absorption values
before and after the reaction with –COOH, and Ms is
the GO samples mass.
Figure 7 represents the result of MB assay that
revealed the carboxylation process successfully in-
creased the amount of COOH groups on the surface of
NGOS. However, there was no significant difference
for OXG0.5 and OXG3 compared to the NGOS. It was
apparent that the 2 M chloroacetic acid generated the
highest amount of COOH resulting from the conver-
sion of OH and epoxide groups. These results are in
agreement with the FTIR and zeta potential data. It is
expected that alkylation process will happen on a
hydroxyl group when using chloroacetic acid. How-
ever, strong basic condition is required for this
nucleophilic substitution in order to significantly
deprotonate the hydroxyl groups. It is speculated that
Page 11 of 15 88
activation of hydroxyl groups under basic condition
(NaOH 4 M) using 2 M chloric acid provided more
proper condition for deprotonation of hydroxyl group
at the same time as enough concentration for a higher
degree of substitution. It is believed that in the lower
concentration (0.5 M) of Cl–C–COOH, inadequate
carboxylation agent resulted in a lower substitution
whereas in higher concentration (3 M) of Cl–C–
COOH interference in de-protonation step caused by
neutralize basic effect may have decreased carboxy-
lation efficiency.
TNBS assay
In order to estimate the best carboxylation condition
for improved bio-conjugation efficacy, octaarginine
(R8) was utilized as a model of amine containing
biomolecules. The R8 peptide is small, highly cationic
peptide which has ability to cross the plasma mem-
brane of eukaryotic cells (Shiroh 2002). It is shown
that the transduction efficacy of different nano-
123
88 Page 12 of 15 J Nanopart Res (2015) 17:88

Fig. 6 Absorption peaks of

the MB solution at different
concentrations a The inset
presents the calibration
curve showing the relation
between absorbance and
concentration at 664 nm.
b Absorption spectra of the
MB solution before and after
interaction with the NGOS–
COOH (OXG2)

particles or biomolecules (e.g. proteins, nucleic acids)
into cells can be improved using R8 peptide because of
its natural-based composition and low cytotoxicity
(Yukawa et al. 2010).
For quantification of the conjugated R8 peptides to
NGOS with different carboxylation ratios, TNBS
assay was utilized. The TNBS would react with
primary amino groups of amino acids in aqueous
solution (pH 8) and form a yellow component which
can be detected at 345 nm. To quantify the peptide
functionalization efficacy, the absorbance value of the
samples after interaction with TNBS solution was read
and compared using a plotted standard curve. The
amount of bonded R8 peptides (lmol/mg) and conju-
gation efficacy are calculated and listed in Table 1. It
can be seen that the amount of bounded peptide is
almost proportional to the concentration of COOH
which is detected on the surface of different NGOS
samples. The maximum R8 peptide conjugation was
obtained for OXG2 sample. The calculated bio-
conjugation efficacy showed 2.53 times increase for
OXG2 rather than NGOS which is used as control. The

123
J Nanopart Res (2015) 17:88 Page 13 of 15 88

Fig. 7 The histogram

presenting the quantitative
measurement of carboxyl
groups in graphite, GO and
its derivatives based on the
MB assay

Table 1 The quantification of conjugated R8 peptide and bio- chloroacetic acid. The samples were then character-
conjugation efficacy on the NGOS-COOH with different car- ized by FTIR, UV–Vis spectroscopy, AFM, XRD, and
boxylation rations based on TNBS assay
zeta potential. The quantitative evaluation of the
Sample Conjugated-R8 Conjugation carboxyl groups was carried out by MB assay. The
(lmol/mg) efficacy %
synthesized nano-carriers with different carboxylation
NGOS 0.080 ± 0.002 5.30 ratios was functionalized by octaarginine peptide
OXG0.5 0.107 ± 0.007 6.60 sequence (R8) as a model. The quantification of
OXG1 0.118 ± 0.011 7.80 attached R8 peptides to graphene nano-sheets’ surface
OXG2 *
0.134 ± 0.009 13.40* is performed with TNBS assay. The results clearly
OXG3 0.12 ± 0.015 8.00 indicated that the reaction between chloroacetic acid
and GO under basic condition occurred successfully
* p value \0.005 compared to NGOS
and most of the unreactive oxygen containing groups
(e.g. hydroxyl, ether and epoxy) was transformed to
result of TNBS assay indicated that by increasing the COOH. The optimum concentration of chloroacetic
amount of COOH groups on the surface of NGOS acid that created the most carboxyl groups was
samples, the bio-conjugation efficacy could be in- determined by MB assay. The resultant NGOS-COOH
creased. As a results, it would be beneficial to optimize solution remained well-dispersed rather than NGOS
the carboxyl functional groups on the surface of which is beneficial for succeeding biological applica-
NGOS samples in order to increase the available sites tions. The TNBS assay confirmed that the NGOS-
for biomolecules attachment and improved bio- COOH sample with optimized carboxylation ratio
conjugation. could be functionalized with R8 peptide 2.5 times
more efficiently compared to the NGOS with no
treatment. In summary, exfoliated GO sheets have
Conclusion found a great potential application for conjugation
with proteins, peptides, nucleic acids, and other
The aim of the present study was to quantitatively biomolecules due to their various surface functional
optimize the carboxylation process for a subsequent groups. Obviously, transformation of an existing
efficient bioconjugation application. In this way, functional group into another group that has a higher
nano-graphene oxide sheets were firstly prepared by conjugational property could serve as useful tool to
modified Hummer method which was followed by efficiently enhance biomolecular conjugation for par-
carboxylation process using various concentrations of ticular application.

123
88 Page 14 of 15
Acknowledgments The authors wish to thank financial
supports provided for this work by the National Institute of
Genetic Engineering and Biotechnology (NIGEB). We also
gratefully acknowledge the scientific help and assistance of
Mohammad Hasan Mohammadzadeh and Mahdi Amrollahi.
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