Hemin-functionalized reduced graphene oxide nanosheets reveal peroxynitrite reduction and isomerization activity

Article abstract of DOI:10.1002/chem.201202272

Facile and efficient reduction of graphene oxide (GO) and novel applications of the reduced graphene oxide (RGO) based materials are of current interest. Herein, we report a novel and facile method for the reduction of GO by using a biocompatible reducing agent dithiothreitol (DTT). Stabilization of DTT by the formation of a six-membered ring with internal disulfide linkage upon oxidation is responsible for the reduction of GO. The reduced graphene oxide is characterized by several spectroscopic and microscopic techniques. Dispersion of RGO in DMF remained stable for several weeks suggesting that the RGO obtained by DTT-mediated reduction is hydrophobic in nature. This method can be considered for large scale production of good quality RGO. Treatment of RGO with hemin afforded a functional hemin-reduced graphene oxide (H-RGO) hybrid material that exhibited remarkable protective effects against the potentially harmful peroxynitrite (PN). A detailed inhibition study on PN-mediated oxidation and nitration reactions indicate that the interaction between hemin and RGO results in a synergistic effect, which leads to an efficient reduction of PN to nitrate. The RGO also catalyzes the isomerization of PN to nitrate as the RGO layers facilitate the rapid recombination of ^.NO₂ with Fe^IV=O species. In the presence of reducing agents such as ascorbic acid, the Fe^IV=O species can be reduced to Fe^III, thus helping to maintain the PN reductase cycle.

Full text of DOI:10.1002/chem.201202272

DOI: 10.1002/chem.201202272
Hemin-Functionalized Reduced Graphene Oxide Nanosheets Reveal
Peroxynitrite Reduction and Isomerization Activity
Amit A. Vernekar and Govindasamy Mugesh*[a]
Abstract: Facile and efficient reduction
of graphene oxide (GO) and novel ap-
plications of the reduced graphene
oxide (RGO) based materials are of
current interest. Herein, we report a
novel and facile method for the reduc-
tion of GO by using a biocompatible
reducing agent dithiothreitol (DTT).
Stabilization of DTT by the formation
of a six-membered ring with internal
disulfide linkage upon oxidation is re-
sponsible for the reduction of GO. The
reduced graphene oxide is character-
ized by several spectroscopic and mi-
croscopic techniques. Dispersion of
RGO in DMF remained stable for sev-
eral weeks suggesting that the RGO
obtained by DTT-mediated reduction
is hydrophobic in nature. This method
can be considered for large scale pro-
duction of good quality RGO. Treat-
ment of RGO with hemin afforded a
functional hemin-reduced graphene
oxide (H-RGO) hybrid material that
exhibited remarkable protective effects
against the potentially harmful peroxy-
nitrite (PN). A detailed inhibition
study on PN-mediated oxidation and
nitration reactions indicate that the in-
teraction between hemin and RGO re-
sults in a synergistic effect, which leads
to an efficient reduction of PN to ni-
trate. The RGO also catalyzes the iso-
merization of PN to nitrate as the
RGO layers facilitate the rapid recom-
bination of NO2 with FeIV =O species.
C
In the presence of reducing agents such
as ascorbic acid, the FeIV =O species
can be reduced to FeIII, thus helping to
maintain the PN reductase cycle.
Keywords: antioxidants · graphene ·
hemin · nanosheets · peroxynitrite
Introduction
been noticed during the use of these reducing agents, which
affect the electrical properties of graphene and their use in
biological and biochemical applications.
Graphene, a single atom thick and 2D material has attracted
significant interest in the areas of materials science and biol-
ogy.[1] The remarkable electronic and mechanical properties,
high surface area and high conductivity led to the use of gra-
phene in nanoelectronics, nanocomposites, nanophotonics,
energy storage, catalysis, biosensors, and drug delivery.[1] To
exploit these attributes, a large scale production of superior
quality graphene sheets is highly desirable. Wide variety of
approaches to produce graphene have been established in
recent literature like chemical vapor deposition (CVD),[2a,b]
micromechanical exfoliation of graphite,[2c] epitaxial
growth,[2d] and liquid-phase exfoliation.[2e] Chemical reduc-
tion of graphene oxide (GO) has marked an impact on the
generation of copious quantity of reduced graphene oxide
(RGO) with cost effectiveness.[3] NaBH4,[3b,c] N2H4,[3d,e] hy-
droquinone,[3f] dimethyl hydrazine,[1b] and Fe/HCl[3g] have
been shown to reduce GO. However, great care is essential
while using these highly toxic and explosive reducing agents.
Furthermore, formation of adducts with the introduction of
other functionalities and metal impurities in RGO have
Functionalization of graphene with polar molecules im-
parts the hydrophilicity over hydrophobic effects, and thus
enhances its dispersibility in polar solvents.[1h] Particularly,
noncovalent functionalization of graphene with biomole-
cules decipher the graphene to be highly biocompatible with
the conservation of intrinsic properties of graphene.[1j]
Hemin, a well-known protoporphyrin found at the active
sites of heme proteins, plays a key role in biochemical reac-
tions and electron-transport chain. Hemin and related met-
alloporphyrins have been shown to exhibit peroxidase-like
and antioxidant activities.[4] Recently, hemin-functionalized
graphene nanosheets have been shown to exhibit peroxidase
activity, to differentiate ss- and ds-DNA,[5a] to oxidize pyro-
gallol effectively,[5b] to selectively and quantitatively detect
cancer cells.[5c] Herein, we describe a new method for the re-
duction GO using dithiothreitol (DTT) as reducing agent.
DTT is a remarkably strong reducing agent (in comparison
to the glutathione, cysteine, and so on) with redox potential
of À0.33 V at pH 7. We also demonstrate, for the first time,
that hybrid nanosheets obtained by functionalization of
RGO with hemin exhibit remarkable peroxynitrite (PN) iso-
merase and reductase-like antioxidant activities.
[a] A. A. Vernekar, Prof. Dr. G. Mugesh
Department of Inorganic and Physical Chemistry
Indian Institute of Science
Bangalore 560 012 (India)
E-mail: mugesh@ipc.iisc.ernet.in
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201202272.
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Results and Discussion
at 230 nm, which could be attributed to the p–p* transition
resulting from C=C bonds of aromatic skeleton. A broad
shoulder in the 290–300 nm region corresponds to the
n–p* transition of C=O bonds from the carboxylic acid func-
tionalities on GO. Reduction of GO using DTT shifts the
maximum absorption from 230 nm to 267 nm due to the re-
storation of conjugation that was distorted in GO. Further-
more, disappearance of the shoulder accounting for the
C=O bond that appeared in GO confirms the deoxygenation
process and removal of carboxylic acid groups. An increase
in the absorbance was also observed due to the reduction
after 60 min at 808C. A similar reduction carried out at
room temperature (288C) resulted in partial reduction of
GO and hence, high temperature was employed in the
study. The reduction was further confirmed by FT-IR analy-
sis. Figure 1B shows the comparative IR spectra of GO and
RGO. A strong absorption band at 1728 cmÀ1 is exhibited
by GO due to the stretching of C=O. A broad band in the
The GO required for the synthesis of RGO was obtained
using the Hummers method.[6] Ultrasonication of GO in
water resulted in homogeneous dispersion of GO, which was
further subjected to the reduction process. DTT as a reduc-
ing agent was then added to the aqueous dispersion of GO.
Reduction process in the neutral conditions led to only par-
tial reduction of GO. DTT appears to be an active reducing
agent in the alkaline pH range due to the formation of reac-
tive thiolate upon deprotonation. Scheme 1 shows the reduc-
region 3600–3200 cmÀ1 corresponding to the O H stretching
À
À
vibrations is also observed along with O H deformation vi-
bration at 1389 cmÀ1. Stretching vibrations at 1618 cmÀ1 and
1043 cmÀ1 are attributed to the C=C and C O units in GO,
Scheme 1. Schematic representation of the synthesis of RGO by the re-
duction using DTT.
À
respectively. In contrast, RGO showed no characteristic ab-
sorption for vibrations from C=O. A dramatic decrease in
À
À
the absorption of C O and O H groups was observed, indi-
cating the effective reduction of GO to RGO.
tion of GO to RGO in the presence of DTT. An enhance-
ment of the reduction rate was observed upon addition of
ammonia as a base in this reaction. This result is consistent
with the earlier report in which facile deoxygenation of GO
has been shown to occur under alkaline conditions.[7] The
strong reducing properties of DTT have been attributed to
its ability to form intramolecular disulfide bond. Although
GO has been shown to oxidize thiols to dilsufides in non-
aqueous media, the route yields carbon materials that are
not nanosheets.[8] Therefore, water appears to be a prefera-
ble medium for the exfoliation of graphite oxide and reduc-
tion of GO to get RGO nanosheets.
Raman scattering is an essential technique to characterize
the structural electronic properties of graphite and graphene
based materials. Chemical oxidation of pristine graphite to
GO followed by the reduction of GO to RGO induces enor-
mous structural changes which could be followed by Raman
spectra. Figure 2a shows the Raman spectra of pristine
Color change of the GO dispersion from brown to black
in the presence of DTT was the preliminary observation that
indicates the formation of RGO (see Scheme 1 and Scheme
S1 in the Supporting Information for a color figure). The
formation of RGO was confirmed by UV/Vis spectroscopy.
Figure 1A shows the UV/Vis spectra of GO and RGO. The
dispersion of GO in water exhibits a maximum absorption
Figure 2. A) Raman spectra and B) XRD patterns of a) graphite, b) GO,
and c) RGO.
graphite, GO and RGO. The G band due to the first order
scattering of the
E
2g phonons of sp2 carbon atoms
(1575 cmÀ1) and D band originating from a breathing mode
of k-point photons of A1g symmetry are the two main char-
acteristic features of graphene based materials.[3g,9] In this
study, the G band for the pristine graphite appeared at
1573 cmÀ1 as the sole characteristic for the first order scat-
tering of the E2g mode. Oxidation of graphite resulted in
broadening and shifting of the G band to 1587 cmÀ1 in GO.
Figure 1. A) UV/Vis absorption spectra and B) FTIR spectra of a) GO
and b) RGO.
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G. Mugesh and A. A. Vernekar
In addition to the G band, a prominent D band is also ob-
served at 1349 cmÀ1, which is ascribed to the destruction of
sp2 character and extensive oxidation induced defects in
sheets. After the reduction of GO by DTT, the Raman spec-
tra of RGO still exhibits D and G bands at 1347 cmÀ1 and
1583 cmÀ1, respectively. The ratio of the intensities of D to
G band (ID/IG) is found to be 0.98 in case of GO. Interest-
ingly, there was a small increase in the ID/IG ratio for RGO
(1.06) upon reduction of GO. The major effect of deoxyge-
nation, restoration of the sp2 network and existence of small
and isolated aromatic domains are responsible for the ob-
served increase in the ID/IG ratio in RGO.[3g,9c,10] It should be
noted that this ratio is lower than those reported for the
RGO produced using NaBH4 (>1),3c N2H4 (1.63),[11] hydro-
quinone (>2),[3f] pre-reduction by NaBH4 (1.91).[12] The
ID/IG ratio is comparable to those obtained for solvothermal
route (1.16),[13] hydrothermal method (0.90),[14] GO/NaBH4/
H2SO4/annealing (0.82).[12] It has been shown recently that
the reduction of GO by Fe/HCl is an effective method re-
sulting in ID/IG ratio equal to 0.32 for RGO obtained after
360 min of reduction time.[3g] A comparison between ID/IG
ratios of RGO obtained via different routes is shown in
Table 1.
Figure 3. Deconvoluted C1s XPS spectra of A) GO B) RGO.
(Figure 3A) clearly indicates the extensive degree of oxida-
À
À
tion. C=C/C C of aromatic rings, C O, and C=O groups
show signals at 284.5, 286.7, and 287.9 eV, respectively.
Upon reduction of GO by DTT, a dramatic decrease in the
intensities of all C1s signals of the oxygen bound carbons
and sp3 carbon (Figure 3B) was observed, suggesting the de-
oxygenation of majority of oxygen containing functional
groups. In addition, the increase in intensity of sp2 carbon
peak indicates that the distortion in conjugation induced
upon oxidation in GO is restored after reduction by DTT.
In this regard, DTT appears to be a much better candidate
for the reduction of GO as compared to the monothiol glu-
tathione.[18]
Table 1. Comparison of ID/IG ratios of RGO obtained by different meth-
ods.
A comparison between the thermal stabilities of GO,
RGO and graphite was also studied by thermogravimetric
analysis (TGA; Figure 4). The TGA profile obtained for
Reducing agent/methods
ID/IG
References
N2H4
NaBH4
1.63
>1
[11]
[3c]
Na+EtOH (solvothermal)
hydroquinone
1.16
>2
[13]
[3f]
l-cysteine
1.2
[15]
dextran
1.03
0.82
0.32
1.44
1.06
[16]
[12]
[3g]
[17]
GO/NaBH4/H2SO4 annealing at 11008C
Fe/HCl
baker’s yeast
DTT
this work
To gain some insights into the structure of RGO, the dis-
tance between the two layers of RGO was calculated. The
X-ray diffraction (XRD) patterns of pristine graphite, GO
and RGO are given in Figure 2B. Pristine graphite exhibits
only a single peak (002) at 2q=26.58 with d spacing=
0.335 nm. Introduction of oxygen functionalities on either
sides of RGO sheets upon oxidation of graphite and interca-
lation of water molecules shifts the 002 reflection peak to
lower angle 2q=11.18 with d spacing=0.796 nm. Upon re-
duction of GO by DTT, the 002 reflection peak of graphite
oxide disappears and the broad peak appears at 2q=24.38
with d spacing=0.366 nm corresponding to the exfoliation
of the layered RGO sheets. To further understand the thick-
ness of layers, atomic force microscopy (AFM) and trans-
mission electron microscopy (TEM) were employed (see
below).
Figure 4. TGA profiles of a) GO, b) RGO, and c) graphite.
GO shows a mass loss of 11% at 1008C, which can be at-
tributed to the loss of adsorbed and intercalated water mole-
cules between GO layers. A significant increase in the
weight loss was observed in the temperature range 170–
2408C due to the removal of thermally labile functional
groups like hydroxyl or epoxy, in the form of CO, CO2, or
water vapor.[19] In contrast, RGO exhibited pronounced
thermal stability due to the prior deoxygenation of labile
functional groups by DTT. A weight loss of 3.5% is shown
by RGO in Ar gas flow at 2008C, which is much less as
compared to that shown by GO. This suggests the presence
of lower amount of oxygen containing functionalities as a
To illustrate the formation of RGO, we further employed
X-ray photoelectron spectroscopy (XPS) to study the deoxy-
genation process. Deconvolution of the C1s spectrum of GO
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Hemin-Functionalized Reduced Graphene Oxide Nanosheets
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result of effective reduction. This loss is comparable to pris-
tine graphite showing 2% weight loss at 2008C. Interesting-
ly, the weight loss at 2008C for RGO is less than that report-
ed for the reduction using monothiols such as glutathione
(ca. 10%),[18] cysteine (ca. 10%),[15] and other reducing
agent such as urea (>10%).[20] This indicates that DTT is a
more efficient reducing agent than the monothiol counter-
parts and urea due to the reactive dithiolates formed in the
reaction and assisted stability of the formation of stable six-
membered ring after its oxidation. A 25% weight loss at
6008C of RGO may be attributed to the pyrolysis of carbon
skeleton, which is similar to that observed for GO and
graphite.
shows a perfect six-fold symmetry as shown in the Fig-
ure 5D. The well-defined diffraction pattern further indi-
cates the crystalline nature of the RGO. Intensity differen-
ces in the diffraction spots suggest the formation of few
layers of RGO due to its intrinsic nature of formation of
stacks.[22]
Atomic force microscopy (AFM) has been used routinely
to study the surface morphology, thickness of sheets and to
identify number of layers of graphene sheets. Figure 6 dis-
The SEM, TEM, and HRTEM images of RGO are shown
in Figure 5. The SEM image (Figure 5A) of solid powder of
RGO on carbon tape reveals the randomly oriented and
Figure 6. AFM image of RGO with cross section showing 0.67 nm thick-
ness of one of the several sheets.
Figure 5. A) SEM, B) TEM, C) HRTEM, D) SAED pattern of RGO
nanosheets. TEM image reveals the corrugation and scrolling of nano-
sheets. Few layers are seen in HRTEM image. The SAED pattern corre-
sponds to image C.
plays the AFM image recorded after casting a sonicated
RGO dispersion drop on freshly cleaved mica surface. The
thickness of RGO has been determined to be 0.67 nm corre-
sponding to a single layer of RGO. This thickness is consis-
tent with that reported earlier for single layer graphene.[23]
To further confirm the formation of RGO, we have car-
ried out 13C MAS NMR study. In the 13C NMR spectra of
GO (Figure 7), the signals at 59.94 and 68.58 ppm can be at-
tributed to 13C nuclei bearing epoxide and hydroxyl groups,
respectively.[23b–e] Graphitic or unoxidized sp2 carbon exhibit-
ed a peak at 130.29 ppm and the carbonyl carbon displayed
its resonance at 190 ppm. A small peak at 166.90 ppm may
arise from O=C-O groups (ester carbonyl) as shown by
Gao et al.[12] Interestingly, all the above signals originating
from the oxygenated and carbonyl carbons disappeared
after the reduction of GO by DTT. The appearance of
broad resonance from 50–150 ppm, thus, confirms the resto-
thin curved sheets. Due to the low resolution limit of SEM
and the use of solid powder sample, this data do not prove
well to view individual sheets of RGO. Therefore, TEM
samples were prepared by casting a drop of dispersion of
RGO in ethanol on a Cu grid. RGO nanosheets were seen
as crumpled silk veil waves as shown in Figure 5B, which is
consistent with earlier report.[3f] Bending of graphene sheets
is a result of thermodynamic stability of the 2D membrane
giving rise to corrugation and scrolling as intrinsic feature of
graphene sheets.[21] The thickness of RGO is found to be
around 1–2 nm consisting of few (2–4 layers) layers stacked
individual graphene sheets due to its inherent scrolling and
folding nature as shown in HRTEM image (Figure 5C). Se-
lected area electron diffraction (SAED) pattern of RGO
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G. Mugesh and A. A. Vernekar
Scheme 2. Proposed mechanism of the reduction of GO by DTT.
The reduction of GO by DTT may follow a mechanism
shown in Scheme 2. The deprotonation of DTT under basic
condition results in the formation of reactive thiolates,
which can behave as potent nucleophiles. In an interaction
of DTT thiolates with GO, the epoxy ring is first opened up
after an attack of one of the thiolates to give intermediate 1.
Due to the thermodynamic stability of the formation of six-
membered ring (DTTox), the rapid cyclization of DTT results
in the reduction of GO. In addition to this, the exothermic
nature of the reaction plays a role in achieving further aro-
maticity by the removal of hydroxyl and carbonyl groups.
The formation of oxidized DTT (DTTox) was confirmed by
HPLC (Figure S1–S3 in the Supporting Information).
Figure 7. Solid-state 13C MAS NMR spectra of A) GO and B) RGO.
ration of sp2 carbons and formation of RGO. This result sup-
ports the data obtained from XRD, XPS, TG.
Owing to the higher propensity of polar groups on GO, it
is highly soluble in water than in organic solvents. Upon re-
duction of GO with DTT, the RGO sheets formed were
well dispersible in DMF than in water confirming the hydro-
phobic nature of RGO (Figure 8A). This dispersion re-
Although RGO is hydrophobic in nature, it can be readily
functionalized to impart hydrophilic behavior and high dis-
persibility in aqueous media.[1h] Non-covalent functionaliza-
tion provides a hybrid material having a high dispersibility
without adversely affecting the intrinsic properties of gra-
phene.[1j] Particularly, functionalization of graphene sheets
with biomolecules not only improves their dispersibility in
aqueous media, but also renders them to be biocompatible
with novel properties originating from the combined effects
of graphene and functionalized molecules. Accordingly,
hemin, a redox active moiety found at the active sites of
heme proteins was stabilized on RGO sheets by noncovalent
functionalization through p--p stacking interactions to yield
H-RGO hybrid nanosheets (Scheme 3).
Figure 8. A) Colloidal dispersion of RGO in DMF exhibits Tyndall effect
upon shining a laser beam. B) Dispersion of RGO prepared after ultraso-
nication for 1.5 h in water, which was stable for few minutes. C) Addition
of salt (NaCl) solution to the RGO dispersion in water led to the imme-
diate precipitation of RGO.
mained stable for several weeks than in water where faster
agglomeration was observed. Furthermore, the RGO disper-
sion in DMF shows a Tyndall effect on shining a red laser
beam (Figure 8A), which confirms the colloidal nature of
RGO dispersion. Similar observations were also reported by
Gao et al.[12] Further, we also studied the effect of salt on
the dispersibility of RGO. Addition of a salt (NaCl) solution
to the dispersion of RGO (Figure 8B) resulted in quick ag-
glomeration of RGO suggesting the efficient removal of
oxygen containing functionalities from GO by DTT (Fig-
ure 8C). This type of salt effect on dispersion of graphene
has also been shown previously by Li et al.[24]
Scheme 3. Schematic representation of the synthesis of H-RGO nano-
sheets.
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Hemin-Functionalized Reduced Graphene Oxide Nanosheets
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the Supporting Information), which indicates the partial sur-
face adsorption of hemin on both the accessible sides of the
RGO layer. The SEM (Figure 9C) and TEM images (Fig-
ure 9D) of H-RGO hybrid showed flaky features with
folded morphologies.
Having synthesized the H-RGO hybrid nanosheets, it was
thought worthwhile to investigate the PN (ONOOÀ) scav-
enging activity of this material. PN is a potent biological oxi-
dizing and nitrating agent that is generated in vivo by a dif-
C
fusion-controlled reaction of nitric oxide ( NO) and superox-
CÀ
ide (O2 ).[27] PN is known to induce DNA modification[28]
and lipid peroxidation in biomembranes.[29] It can oxidize
protein/non-protein thiols[30] and inactivate enzymes by ni-
tration of tyrosine residues, which affect the signal transduc-
tion.[31,32] Although several small molecules such as ascor-
bate, methionine, cysteine, thioureas, selenium compounds,
and metalloporphyrins have been shown to scavenge PN,[33]
the effect of graphene-based nanomaterials on PN-mediated
oxidation or nitration have not been investigated. Therefore,
the inhibition of PN-mediated oxidation of dihydrorhoda-
mine to rhodamine 123 was studied by fluorescence spectro-
scopy. The IC50 values (the amount of materials required to
inhibit 50% of the oxidation) were obtained by plotting
the% oxidation of DHR versus the amount of scavengers
(Figure 10).
Figure 9. A) UV/Vis spectra of a) hemin, b) H-RGO, and c) RGO.
B) Peaks of Fe2P in XPS of H-RGO. C), D) SEM and TEM images of
H-RGO, respectively.
The non-covalently grafted hemin on RGO was character-
ized by various methods. The UV/Vis spectrum (Figure 9A)
reveals a bathochromic shift of soret band of hemin from
385 nm to 418 nm with very weak Q bands. The large batho-
chromic shift of 33 nm is observed due to p--p stacking in-
teraction between graphene and hemin as reported in the
literature.[5a] The estimated amount of hemin on RGO was
found to be 1.42% as calculated by comparing the absorb-
ance of hemin and H-RGO after the blank correction with
RGO. Owing to the noncovalent interactions between RGO
and hemin in H-RGO, the D and G bands are shifted slight-
ly to 1352 cmÀ1 and 1590 cmÀ1, respectively as observed
from Raman spectra of H-RGO (Figure S4 in the Support-
ing Information). There is no apparent change in the ID/IG
ratio of H-RGO (1.04) and RGO (1.06), thus indicating the
conservation of conjugation upon functionalization of RGO
with hemin as reported elsewhere for Picket–Fence porphyr-
in functionalized graphene hybrid.[25] Upon cyclic voltamme-
try, H-RGO nanosheets exhibit redox peaks across the
equivalent cathodic and anodic current intensities with
formal potential [Eo’] equal to À0.44 V (vs. Ag/AgCl in satu-
rated KCl) and peak to peak separation of 76 mV. On the
other hand, bare glassy carbon electrode, RGO in N2 satu-
rated phosphate buffer (pH 7.4) did not show any such
peaks (Figure S5 in the Supporting Information). X-ray pho-
toelectron spectrum (XPS) of H-RGO shows peaks at
711.5 eV and 398.6 eV (Figure 9B), which can be attributed
to Fe2p and N1s of hemin on H-RGO nanosheets with
1.35% loading of hemin on RGO (Figure S6 in the Support-
ing Information). The binding energy of Fe2p in hemin
shows a shift from the reported values for hemin by approx.
1 eV, thus suggesting the existence of strong electronic inter-
actions between hemin and RGO.[26] AFM image reveals the
thickness of H-RGO nanosheets to be 1.2 nm (Figure S7 in
Figure 10. A) PN scavenging activity in PN-mediated oxidation of DHR
to rhodamine 123 by scavengers. B),C) Activity plots of oxidation of
DHR in the presence of H and H-RGO, respectively.
Interestingly, a remarkable PN scavenging activity with an
IC50 value of 2.15Æ0.19 mg was observed for the H-RGO
hybrid nanosheets in which the hemin content was only
about 30.44 ng. In contrast, the IC50 value obtained for
hemin in its isolated form was 2.54Æ0.40 mg, which indicates
that the PN scavenging activity of hemin is increased by
about 84-fold upon functionalization with RGO. These ob-
servations suggest that RGO acts synergistically with hemin
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G. Mugesh and A. A. Vernekar
in exhibiting the PN scavenging activity. This is probably
due to the p donor property of graphene to the Fe center of
hemin through cationic p interactions.[5b] Such a synergistic
effect has been demonstrated previously for a selective and
quantitative cancer cell detection using folic acid conjugated
graphene–hemin composite[5c] and oxidation of pyrogallol.[5b]
In contrast to the H-RGO hybrid, RGO alone did not show
any noticeable scavenging activity at lower concentrations.
However, a significant decrease in the fluorescence intensity
was observed at higher concentrations, thus indicating that
RGO may quench fluorescence at higher concentrations.[34]
In contrast to H-RGO nanosheets, GO and GO-hemin mix-
tures did not show any significant effect on the PN-mediated
oxidation of DHR. These observations suggest that the
p--p stacking interactions between the RGO and hemin play
a significant role in the observed antioxidant effect.
In addition to the decomposition of PN in PN-mediated
oxidation, we have also investigated the effect of H-RGO
nanosheets on PN-mediated nitration by using free l-tyro-
sine by UV/Vis spectroscopy. The nitration of tyrosine has
attracted immense interest in biology as nitrotyrosine has
been extensively used as a potential biomarker for oxidative
and nitrosative stress.[35] Furthermore, the formation of 3-ni-
trotyrosine has been implicated in different pathophysiologi-
cal states such as neurological disorders, multiple sclerosis,
atherosclerosis, Parkinsonꢁs disease, viral infections, rejec-
tion of transplanted organ, ischemia reperfusion injury.[36]
Nitration of protein-bound tyrosyl residue is also known to
inactivate enzymes such as mitochondrial ATPase, tyrosine
hydroxylase, tyrosine kinase, a-thrombin, cytochrome P450,
manganese superoxide dismutase.[32b,37] In the present study,
when free hemin was tested as a scavenger of PN in PN-
mediated nitration of free l-tyrosine, no scavenging activity
was observed (Figure 11). In fact, an increase in the forma-
tion of 3-nitro-l-tyrosine upon supplement of 1 mg to 50 mg
hemin was observed, indicating that free hemin catalyzes
the formation of 3-nitro-l-tyrosine. Interestingly, when
H-RGO was employed as a scavenger, the IC50 value of
2.50Æ0.04 mg with hemin content of 35.40 ng was obtained.
The PN scavenging properties of H-RGO nanosheets in the
nitration reactions are similar to that observed for the oxi-
dation reaction. As mentioned earlier, synergistic effect of
H-RGO plays a major role in scavenging of PN in these po-
tentially harmful PN-mediated reactions.
Figure 11. A) PN scavenging activity in PN-mediated nitration of free
l-tyrosine to 3-nitro-l-tyrosine. B) Activity plots of nitration of free l-ty-
rosine in the presence of various concentrations of H-RGO.
To understand whether the H-RGO nanosheets can scav-
enge PN in PN-mediated nitration of tyrosine residues in
proteins, we investigated the inhibition of PN-mediated ni-
tration of bovine serum albumin (BSA). As BSA contains
several tyrosyl residues that could be targets for nitration,
the PN-mediated nitration of this protein has been studied
extensively. The H-RGO hybrid nanosheets exhibited 70%
scavenging activity in the PN-mediated nitration reaction as
seen from Figure 12. In contrast, hemin and RGO showed
only 6.90Æ0.21% and 21.49Æ0.92% scavenging activity, re-
spectively (Figure 12). As the oxidation of the FeIII center in
heme is expected to produce the corresponding FeIV-oxo
Figure 12. A) PN scavenging activity in PN-mediated nitration of tyrosyl
residue in BSA by scavengers. B) Anti-nitrotyrosine immunoblotting of
BSA after treatment with PN in the absence and presence of scavengers.
H-RGO having 1.13% of hemin content was used.
achieved by reducing the FeIV center in the oxidized
H-RGO to FeIII. Therefore, we have studied the effect of as-
corbic acid (Asc), a reducing agent, on PN-scavenging activ-
ity of H-RGO nanosheets. When an excess amount of Asc
was added, the scavenging activity of hemin and H-RGO
nanosheets was enhanced by approximately 10 and 12%, re-
À
species, a catalytic reduction of ONOOÀ to NO2 can be
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Hemin-Functionalized Reduced Graphene Oxide Nanosheets
FULL PAPER
spectively. However, there was no significant enhancement
in the activity when only Asc was added to RGO. These ob-
servations indicate that PN reacts with the FeIII center in
H-RGO to produce the corresponding FeIV-oxo species and
À
the conversion of PN to NO2 becomes catalytic in the pres-
ence of Asc. The efficient isomerization and catalytic reduc-
tion of PN by H-RGO can be ascribed to the p–p stacking
interactions between RGO and hemin. The ability of RGO
to attract nitrogen oxides may also contribute to the en-
hanced activity of H-RGO nanosheets.[38] In the absence of
RGO, hemin has a tendency to form stable oxo or peroxo
dimers on reaction with peroxynitrite or other oxidizing
agents.[39] In the case of H-RGO hybrid nanosheets, the
p–p stacking may disfavor the formation of stable oxo and
peroxo dimmers. Furthermore, the H-RGO hybrid can be
Scheme 4. Proposed mechanism for the isomerization and reduction of
PN and scavenging of ·NO2 by H-RGO hybrid nanosheets. Asc=ascorbic
acid.
À
considered as a catalyst for the isomerization of PN to NO3
as these nanosheets having only 350 ng of hemin content
prevents nitration by almost 70% nitration even in the pres-
ence of very high (1800 mm) concentration of PN.
It is known that hemin can enhance the nitration of tyro-
presence of ascorbic acid, the reduction of FeIV=O inter-
À
sine residues in proteins by NO2 and H2O2.[40] In agreement
mediate can also regenerate the FeIII with the elimination of
À
with this, a significant increase in the tyrosine nitration was
observed when free hemin was added to BSA, NaNO2, and
H2O2 (Figure S8 in the Supporting Information). In contrast,
a marginal decrease in the nitration with respect to the con-
trol was observed in the presence of RGO. Interestingly, a
further decrease in the nitration was observed when H-
RGO was used instead of RGO. These observations indicate
that the p–p stacking interactions between RGO and hemin
significantly alter the redox properties of hemin. It should
be noted that heme peroxidases such as myeloperoxidase
NO2 as shown in Scheme 4. The major effect of graphene is
C
the minimization of the NO2 cage escape reaction that is
generally observed for free heme and heme proteins. It
C
should be noted that the rapid diffusion of NO2 from the
heme site is responsible for the tyrosine nitration.
The changes between the two redox states of heme can be
studied by UV/Vis spectroscopy. When PN (1000 mm) was
added to H-RGO (30 mg) in assay buffer (pH 7.4), immedi-
ate decrease in the intensity of soret band of hemin in
H-RGO was observed, suggesting the formation of FeIV =O
species with relative depletion of FeIII species (Figure S9 in
the Supporting Information). After about 50 s of reaction
time, a further increase in the absorbance due to soret band
was noted, which indicates that the FeIV =O species is re-
duced back to FeIII. A possible reason for the change in the
À
(MPO) and horseradish peroxidase (HRP) utilize NO2 and
H2O2 as substrates to catalyze tyrosine nitration in pro-
teins.[41] Recently, Guo et al. have shown that hemin-gra-
phene nanosheets can catalyze the oxidation of different
peroxidase substrates such as 2,2’-azinobis(3-ethylbenzothio-
zoline)-6-sulfonic acid (ABTS), and 3,3’,5,5’-tetramethylben-
zidine (TMB) and o-phenylenediamine (OPD) in the pres-
C
oxidation state is the recombination of NO2 with the
FeIV =O species, which generates nitrate (isomerization
mechanism). The oxidation of the FeIII species with H2O2
(1000 mm) shows a decrease in absorbance of soret band due
to the formation of the FeIV =O species (Figure S10 in the
Supporting Information). When excess NaNO2 (2000 mm)
was added to the reaction mixture containing H-RGO
(30 mg) and H2O2 (1000 mm), an increase in absorbance was
noted after few seconds (Figure S11 in the Supporting Infor-
mation). This increase is probably due to the reduction of
FeIV =O to FeIII with the generation of nitrate.
ence of H2O2.[5a] However, in the presence of NO2 and
À
H2O2, the heme in H-RGO hybrid appears to behave differ-
ently from that of heme peroxidases.
The mechanism for the reduction/isomerization of PN by
H-RGO nanosheets appears to be similar to the one pro-
posed for an iron–porphyrin complex.[4b] According to this
mechanism (Scheme 4), the FeIII center in the H-RGO
reacts with PN to generate FeIII-O-ONO species. The ho-
À
molysis of the O O bond in this species leads to the forma-
tion of caged radical FeIV =O NO2 intermediate. As men-
C
tioned earlier, the RGO layers may help in increasing the
local concentration of nitrite and decreasing their escape,
which may facilitate the recombination of FeIV =O and ni-
trite. It has been shown previously that graphene-based
Conclusion
In this article, a facile and efficient approach for the synthe-
sis of RGO by the reduction of GO using DTT has been de-
scribed. The strong reducing ability of DTT is attributed to
the generation of reactive dithiolates in reaction and ther-
modynamic stability of disulfide state after the reduction of
GO. The reduction by DTT is very efficient than that ach-
nanocomposites can be used as selective sensors for nitrite
IV
anions.[42] Therefore, the recombination of Fe =O and NO2
C
generates the FeIII-nitrato complex. The cleavage of the
III
À
À
Fe O bond in the Fe ONO2 species regenerates the
FeIII center with an elimination of nitrate (NO3À). In the
Chem. Eur. J. 2012, 18, 15122 – 15132
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www.chemeurj.org
15129

G. Mugesh and A. A. Vernekar
finally dried in air. GO obtained was subjected to ultrasonication for
40 min (20% amplitude) in order to exfoliate into graphene oxide in ul-
trapure water. Homogeneous dispersion of GO (1 mgmLÀ1) obtained
was directly used for reduction to get RGO nanosheets.
ieved using glutathione and cysteine. Spectroscopic studies
of RGO confirm the effective removal of oxygen containing
functionalities in GO. Microscopic examination reveals the
formation of few (2–4) layers of RGO (due to corrugation
and scrolling) with pronounced crystallinity. The reduction
of GO by DTT appears to be a good method for the prepa-
ration of good quality RGO in good yield. Noncovalent
functionalization of RGO with hemin resulted in material
that exhibits a remarkable antioxidant activity under physio-
logically relevant conditions. The stabilization of monomeric
hemin on RGO provides the required synergistic effect for
Preparation of RGO: Reduction of GO was achieved by using DTT. Typ-
ically, 200 mg DTT was added to 100 mL of homogeneous GO dispersion
(1 mgmLÀ1). The pH of this dispersion was maintained above 9 by the
addition of 30% ammonia solution and stirred vigorously for 10 min at
308C. Then, the temperature was raised to 808C and maintained for 1 h.
The brown dispersion of GO changed to black in color upon reduction
with DTT. The RGO was centrifuged (8000 rpm) and washed several
times with ultrapure water and finally with ethanol and then dried in air.
Synthesis of H-RGO hybrid nanosheets: Hemin (2 mg) was firstly dis-
solved in 10 mL ethanol by the addition of 80 mL NH3 to get dark red-
dish-brown solution. To this, 40 mg of RGO was added and the reaction
mixture was sonicated for 5 min to ensure the complete dispersion of
RGO. Stirring was continued at room temperature for 6 h. Centrifugation
at 12000 rpm for 15 min resulted in separation of precipitate, which was
washed five times with ammoniated ethanol to remove excess hemin and
finally with ethanol to yield H-RGO nanosheets.
C
an efficient isomerization and reduction of PN. The NO2
scavenging action of H-RGO nanosheets together with the
recombination of NO2 and FeIV =O to form nitrate contrib-
ute to the observed antioxidant effect.
Preparation of RGO and H-RGO modified electrode: The RGO and
H-RGO modified electrode was prepared by a method described else-
where.[5a] The glassy carbon electrode (3 mm) was polished with alumina
slurry, rinsed with ultrapure water. Further, it was washed successively
with 1:1 nitric acid, acetone, and ultrapure water in an ultrasonic bath
and dried in air. The RGO and H-RGO modified glassy carbon electro-
des were obtained by casting a drop of 5 mgmLÀ1 suspension (methanol)
on the surface of electrode, which were dried in air. Finally, the modified
electrodes were activated by several successive scans with a scan rate of
50 mVsÀ1 in phosphate buffer solution (pH 7.4) until a steady voltammo-
gram was obtained.
Experimental Section
Chemicals: Graphite (2–15 mm) and DTT were purchased from Alfa
Aesar and used as received. Sulfuric acid, hydrochloric acid, and potassi-
um permanganate were purchased from S. D. Fine chemicals. Hydrogen
peroxide used in the study was purchased from Merck. Tyrosine used in
the nitration experiments was obtained from Calbiochem. BSA, sodium
nitrite and isoamyl nitrite were purchased from Sigma–Aldrich.
Characterization methods: Absorption spectra were recorded on
Perkin–Elmer Lambda 750 UV/Vis spectrometer. IR spectra were ob-
tained on a Bruker IR spectrometer. Raman spectroscopy was performed
a
Synthesis of peroxynitrite (PN): Peroxynitrite was synthesized by follow-
ing the literature method with minor modifications.[43] A solution of 30%
(ca. 8.8m) H2O2 was diluted to 50 mL with water, cooled to about 48C in
an ice/water mixture, added to NaOH (5N, 30 mL), and diethylene tria-
mine pentaacetic acid (DTPA; 0.04m, 5 mL) in NaOH (0.05 N) with
gentle mixing, and then diluted to a total volume of 100 mL. The concen-
tration of H2O2 in the final solution was 0.5m; the pH ranged from 12.5
to 13.0. The buffered H2O2 was stirred vigorously with an equimolar
amount of isoamyl nitrite (0.05m or 6.7 mL) for 3–4 h at room tempera-
ture. The reaction was monitored by withdrawing aliquots at an interval
of 15 or 30 min and assaying for peroxynitrite at 302 nm using UV/Vis
spectrophotometer. When the yield of peroxynitrite reached a maximum,
the aqueous phase was washed with dichloromethane, chloroform, and
hexane (3ꢃ100 mL) in a separating funnel to remove the contaminating
isoamyl alcohol and isoamyl nitrite. The unreacted H2O2 was removed by
passing the aqueous phase through a column filled with granular MnO2
(25 g). The concentration of the stock solution of peroxynitrite was meas-
ured after 500 times dilution with a NaOH solution (0.1 N) and then as-
saying for peroxynitrite at 302 nm (e=1670mÀ1 cmÀ1) using the UV/Vis
spectrophotometric method.
on
a HORIBA JOBIN YVON LabRAM HR Raman spectrometer.
Powder XRD was recorded on PANalytical Xpert pro theta-two theta
diffractometer using a CuKa (1.5406 ꢂ) radiation. CV was performed on
EG and G PAR Model 253 Versa stat/potentiostat/galvanostat with elec-
trochemical analysis software 270. A three-electrode system was used in
the experiment with a bare and the modified glassy carbon electrode
(3 mm diameter) as the working electrode, respectively. An Ag/AgCl
electrode (saturated KCl) and a Pt wire electrode were used as a refer-
ence and counter electrode, respectively. X-ray photoelectron spectrosco-
py (XPS) was carried out on a MULTLAB 2000 THERMO SCIENTIF-
IC, UK. Thermogravimetric analysis (TGA) was carried out on
a
NETZSCH TG 209 F1 instrument at a heating rate of 28CminÀ1 from
40–7508C. Solid state 13C magic-angle spinning (MAS) NMR spectra
were obtained from 300 MHz Bruker Avance solid state NMR spectrom-
eter using standard Bruker pulse programs. Atomic-force microscopy
(AFM) measurements were performed using Nanoscope V multimode
atomic force microscope (Veeco Instruments, USA) operating in the tap-
ping mode. Scanning electron microscopy (SEM) images were recorded
on Fei Sirion UHR SEM. Transmission electron microscopy (TEM)
images and SAED pattern were recorded on Fei Tecnai T20 Ultra Twin
operating at 200 kV after casting a drop of RGO dispersion in ethanol
over Cu grid.
PN scavenging activity in PN mediated oxidation of dihydrorhodamine:
PN-mediated oxidation of dihydrorhodamine (DHR) was studied using
fluorescence spectroscopy. Fluorescence intensity was measured with ex-
citation and emission wavelengths of 500 nm and 526 nm, respectively.
The stock solution of DHR in dimethylformamide was purged with nitro-
gen and stored at À208C. The working solutions of DHR and PN were
kept on ice bath. The assay mixture contained DHR (0.50 mm), PN
(0.95 mm) in 100 mm phosphate buffer of pH 7.4 and variable inhibitor
concentrations. The fluorescence intensity from the reaction of DHR
with PN was set as 100% and the intensity after the addition of various
scavengers was expressed as the percentage of the intensity observed in
the absence of scavengers. The final fluorescence intensities were correct-
ed for background reactions. The activity plots were obtained using
Origin 6.1 software utilizing sigmoidal curve fitting and these plots were
used for the determination of IC50 values.
Preparation of GO: The graphite oxide was synthesized from graphite
powder following the Hummers method.[6] Typically, concentrated H2SO4
(69 mL) was added to a mixture of graphite powder (3.0 g) and NaNO3
(1.5 g) and the mixture was cooled to 08C. KMnO4 (9.0 g) was added
slowly in portions to keep the reaction temperature below 208C. The re-
action was warmed to 358C and stirred for 30 min, at which time water
(138 mL) was added slowly, producing a large exotherm to 988C. Exter-
nal heating was introduced to maintain the reaction temperature at 988C
for 15 min, then the heating was removed and the reaction was cooled
using water bath for 10 min. Additional water (420 mL) and 30% H2O2
(3 mL) were added, producing another exotherm and to produce bright
yellow precipitate. This mixture was cooled to room temperature and
centrifuged at 4000 rpm. The yielded brownish-yellow precipitate of GO
was washed several times with 5% HCl and then with water/ethanol and
PN scavenging activity in PN-mediated nitration of free l-tyrosine: PN-
mediated nitration of free l-tyrosine was studied using UV/Vis spectro-
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Hemin-Functionalized Reduced Graphene Oxide Nanosheets
FULL PAPER
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5 min before recording absorbance. The formation of 3-nitro-l-tyrosine
was monitored at the wavelength 440 nm. The activity plots were ob-
tained using origin 6.1 software utilizing sigmoidal curve fitting and then
plots were used for the determination of IC50 values.
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Received: June 27, 2012
Published online: October 5, 2012
15132
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ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2012, 18, 15122 – 15132