Cobalt phthalocyanine tetracarboxylic acid modified reduced graphene oxide: A sensitive matrix for the electrocatalytic detection of peroxynitrite and hydrogen peroxide

Article abstract of DOI:10.1039/c4ra09781e

The quantification of peroxynitrite (ONOO^-, PON) and hydrogen peroxide (H₂O₂) is intrinsically difficult as both species show similar oxidative features located within a narrow potential. The sub-second lifetime of ONOO^- at neutral pH further complicates the analysis. In this paper, we examine the electrocatalytic activity of cobalt phthalocyanine tetracarboxylic acid (CoPc-COOH) loaded reduced graphene oxide (rGO) films towards peroxynitrite and hydrogen peroxide detection. The rGO/CoPc-COOH matrix is synthesized by the reaction of graphene oxide (GO) and CoPc-COOH at 90 °C for 5 h under ultrasonication. The integration of CoPc-COOH and the reduction of GO to rGO was confirmed by X-ray photoelectron spectroscopy, FTIR, Raman, UV-vis spectroscopy and electrochemistry. The rGO/CoPc-COOH film showed high electrocatalytic activity and specificity for ONOO^- at anodic potential with a sensitivity of ≈11.5 ± 1 nA nM^-1 and a peroxynitrite detection limit of ≈1.7 nM. The rGO/CoPc-COOH films further exhibited electrocatalytic reduction of H₂O₂ with a sensitivity of 14.5 μA mM^-1 and a detection limit of ≈60 μM for H₂O₂. This journal is

Full text of DOI:10.1039/c4ra09781e

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Table of content
The electrocatalytic properties of cobalt phthalocyanine modified reduced graphene oxide for
peroxynitrite and hydrogen peroxide are investigated.
1

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Cobalt phthalocyanine tetracarboxylic acid modified reduced graphene oxide: a sensitive matrix
for the electrocatalytic detection of peroxynitrite and hydrogen peroxide
1,2#
Ioana S. Hosu, Qiang Wang,
1,3 #
4
Alina Vasilescu, Serban F. Peteu, * Valentin Raditoiu, Svetlana
2
2
1
,5
5
Railian, Vladimir Zaitsev, Kostiantyn Turcheniuk, Qi Wang, Musen Li, Rabah Boukherroub,
1
3
3
1
1
Sabine Szunerits *
1
Institut de Recherche Interdisciplinaire (IRI, USR 3078), Université Lille1,
Parc de la Haute Borne, 50 Avenue de Halley, BP 70478, 59658 Villeneuve d’Ascq, France
2
National Institute for R&D in Chemistry and Petrochemistry,
Spl. Independentei 202, 060021 Bucharest, Romania
3
Key Laboratory for Liquid-Solid Structural Evolution and Processing of Materials,
Shandong University, Jinan 250061, China
4
International Center of Biodynamics, 1B Intrarea Portocalelor, 060101, Bucharest, Romania
5
Taras Shevchenko University, 60 Vladimirskaya str., Kiev, Ukraine
Abstract
-
The quantification of peroxynitrite (ONOO , PON) and hydrogen peroxide (H
2
O
2
) is intrinsically
difficult as both species show similar oxidative features located within a narrow potential. The sub-
-
second lifetime of ONOO at neutral pH complicates furthermore the analysis. In this paper, we
examine the electrocatalytic activity of cobalt phthalocyanine tetracarboxylic acid (CoPc-COOH)
loaded reduced graphene oxide (rGO) films towards peroxynitrite and hydrogen peroxide detection.
The rGO/CoPc-COOH matrix is synthesized by the reaction of graphene oxide (GO) and CoPc-COOH
at 90 °C for 5 h under ultrasonication. The integration of CoPc-COOH and the reduction of GO to
rGO was confirmed by X-ray photoelectron spectroscopy, FTIR, Raman, UV-vis spectroscopy and
electrochemistry. The rGO/CoPc-COOH film showed high electrocatalytic activity and specificity for
#
The two authors have made equal contributions.
*
Authors to whom correspondence should be sent: Sabine Szunerits (Sabine.Szunerits@iri.univ-
lille1.fr); Serban Peteu (serbanfpeteu@gmail.com)
2

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-
-1
ONOO at anodic potential with a sensitivity of ≈11.5 ± 1 nA nM and a peroxynitrite detection limit
of ≈ 1.7 nM. The rGO/CoPc-COOH films exhibited furthermore electrocatalytic reduction of H
O
2 2
-1
2 2
with a sensitivity of 14.5 µA mM and a detection limit of ≈ 60 µM for H O .
Keywords: reduced graphene oxide, cobalt phthalocyanine tetracarboxylic, peroxynitrite, biosensor,
selectivity, nitrite
3

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1. Introduction
Metallophthalocyanines (MPcs) are transition metal complexes with several characteristic properties
that contribute in a major way to their extraordinary versatility. Actual applications include:
electrochromic devices, components in analytical devices or electrocatalytic and photocatalytic
1
2-7
processes. Most of the applications rely critically upon the redox properties of MPcs. Due to their
macrocyclic nature with extended π-systems, phthalocyanines are capable of undergoing fast redox
8
processes. The central metal ion may be inert to redox processes in the usual electrochemical regimes
for transition metal species such as Ni(II) or may be a transition metal such as Fe(II) that undergoes a
redox process at potentials comparable to the phthalocyanine ring processes. Most unsubstituted MPc
species have only very limited solubility in virtually all solvents, thereby limiting solution phase redox
9
measurements. Ring substitution and good choice of the right metal ion center have proven to be
1
0, 11
efficient procedures for rendering MPcs water soluble.
One research area where MPcs have found a wide range of applications is their use as electrocatalysts
in electrochemical sensors. Electrodes modified with phthalocyanine-based macrocycles have shown
1
to be outstanding electrocatalytic sensors for a variety of analytes, including oxygen hydrogen
2
1
3
peroxide, peroxynitrite, nitrite,
14
3, 15, 16
17
7
1
nitrate, nitric oxide (NO) and others. We have
demonstrated that hemin, a protoporphyrin with an iron center, allows for a sensitive and selective
-
.
18-20
detection of ONOO
Next to iron, cobalt is widely used as transition metal in phthalocyanine
complexes (CoPc), where the catalytic behavior is related to Co(II)/Co(III) or Co(II)/Co(I) redox
2
1-23
couples.
One major limitation of electrodes modified by physically absorbed CoPc is that the
complex is detaching from the electrode surface with time and that the complex has relatively low
conductivity. Host matrixes forming stable electrochemical active electrode inerfaces are thus
required. The unique electrical and electrochemical properties of reduced graphene oxide (rGO) makes
2
3-25
this material an ideal host for CoPc complexes.
Immobilization of cobalt phthalocyanine onto
graphene and graphene oxide derivatives has been reported to result in bioelectrochemical platforms
2
for the electrooxidation of L-cysteine, electrochemical reduction of tert-butylhydroperoxide,
5
23
2
6
24
detection of glucose or the oxidation of nitrite. Yang et al. investigated recently the
adsorption/intercalation of cobalt phthalocyanine onto/into graphene oxide layer and studied in detail
2
2
the interaction between GO and CoPc.
We examine in this paper the electrocatalytic activity of cobalt phthalocyanine tetracarboxylic acid
(
(
CoPc-COOH) (1) loaded reduced graphene oxide (rGO) films (Figure 1A) towards peroxynitrite
-
ONOO
,
2 2
PON) and hydrogen peroxide (H O ) detection, for the first time. The electrochemical
-
detection of ONOO and H O are complicated by the fact that both species show similar oxidative
2
2
2
features located within a narrow potential range. The desire to study the key roles of both analytes in
7
cellular signal transduction necessitates the development of an alternative sensing strategy. In addition,
4

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the accurate and rapid determination of hydrogen peroxide is of practical importance in various fields
such as food, pharmaceutical, clinical, industrial and environmental analysis. A sensitive hydrogen
peroxide sensor is also of prime interest for enzyme-based biosensing as hydrogen peroxide is the
2
product of most oxidase enzyme reactions. The use of CoPc-COOH loaded rGO matrixes seems to
8
be a step into these directions. This article discusses the fabrication of this matrix together with its
-
sensitivity towards ONOO and H
2
O
2
.
2
. Experimental part
.1. Materials
2
Graphite powder (<20 micron), hydrogen peroxide (H
SO ), dimethylsulfoxide (DMSO), potassium chloride (KCl), hydrazine monohydrate, sodium
hydroxide (NaOH), ethanol, dimethyl formamide (DMF), ammonium chloride (NH Cl), ammonium
molybdate [(NH MoO ], hydrochloric acid (HCl, 37%), urea, cobalt acetate tetrahydrate, trimellitic
anhydride, potassium hydroxide (KOH), 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS),
tetrabutylammonium tetrafluoroborate (TBATBF ), CAPS buffer (pH 10), and manganese dioxide
MnO ) were purchased from Aldrich and used as received. Alumina (0.05 µm) and diamond (1 µm)
2 2 2
O ), sodium nitrite (NaNO ), sulfuric acid
(
H
2
4
4
4
)
2
4
4
(
2
polishing paste were purchased from ALS, Japan. Glassy carbon electrodes (5 mm in diameter),
platinum wire counter electrode and silver/silver chloride reference electrode were obtained from
Cambria Scientific.
2.2. Synthesis of cobalt tetracarboxyl phthalocyanine CoPc-COOH (1)
Cobalt phthalocyanine tetracarboxylic acid (CoPc-COOH) was prepared in two steps similar to
2
9-31
previous work.
A mixture of trimellitic anhydride (5 g), cobalt acetate tetrahydrate (3.24 g),
ammonium chloride (1g), ammonium molybdate (0.5 g) and urea (10g) was grounded in a ceramic
mortar, transferred to a ceramic crucible and irradiated in a commercial microwave oven (EG 1031NP,
1000 W/2.45 GHz, Hyundai Guangdong, China) at 350 W power for 15 min mixing occasionally to
homogenize the reaction mass. After reaction completion, the formed cobalt phthalocyanine
3
tetracarboxamide was hydrolyzed by heating at 100°C in an aqueous KOH solution (10 g, 90 cm ) for
600 min. The cobalt phthalocyanine tetracarboxylic acid (1) was formed, precipitated through the
addition of HCl (37 wt %) and separated by filtration. All the inorganic compounds eventually present
in the final product were removed by extraction in a Sohxlet apparatus using pure water. Yield: 62%
1
after purification, H-NMR (400 MHz, DMSO-d
6
): δ 78.84-8.05 (m, 1H), 8.28 (s, 1H), 8.88-8.92 (m,
3
H), 11.51 (br., s, 1H –COOH) .
1
1
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2.3. Preparation of CoPc-COOH functionalized reduced graphene oxide (rGO/CoPc-COOH)
3
2
Graphene oxide (GO) was synthesized from graphite powder by a modified Hummers. GO (1
mg/mL) in DMF was mixed with CoPc-COOH (0.2-1 mg/mL) dissolved in DMF and sonicated (464
W, 37 Hz) for 5 h at 90 °C. The precipitate formed at the end of the reaction was separated from the
supernatant by centrifugation for 45 min at 14.000 rpm, washed three times with water and dried at
60°C for 12 h.
2.4. Preparation of reduced graphene oxide using hydrazine
In a typical procedure, hydrazine hydrate (0.50 mL, 32.1 mM) was added to 5 mL of the yellow-brown
GO aqueous suspension (0.5 mg/ mL) in a round bottom flask and heated in an oil bath at 100 °C for
24 h. During this time, the reduced GO gradually precipitated out of the solution. The product was
isolated by filtration over a polyvinylidene difluoride (PVDF) membrane with a 0.45 ꢀm pore size,
washed copiously with water (5×20 mL) and methanol (5×20 mL), and dried in the oven at 60°C for
3
3
6
h .
-
.5. Generation of peroxynitrite (ONOO )
2
2.5.1. From sodium nitrite
3
4
Peroxynitrite (PON) was synthesized as previously described. Briefly, a beaker containing sodium
nitrite (1 M) is kept on ice with vigorous stirring. An ice-cold solution of hydrogen peroxide (1.2 M )
in hydrochloric acid (0.1 M) was added rapidly, followed by ice-cold sodium hydroxide (7 M) to
quench the reaction. Excess hydrogen peroxide was removed by passing the solution over manganese
dioxide flakes. The obtained alkaline stock solution of peroxynitrite (pH =13) was kept in the dark at -
20°C and used within a week. The concentration of the peroxynitrite stock solution was determined
-1
-1 35
prior to each experiment by measuring the UV-Vis absorbance at 302 nm (ε=1670 L mol cm ).
2.5.2. From 3-morpholino-syndnominne (SIN-1)
3-Morpholino-sydnonimine (SIN-1, stored at -20°C) was used for the generation of peroxynitrite for
each experiment; the stock solution was simply formed by mixing SIN-1 with deoxygenated PBS at
pH 7.4 and room temperature. The SIN-1 complex releases both nitric oxide and superoxide in
solution, in the presence of atmospheric oxygen with a 1/1 stoichiometry, and after reaching steady
6

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3
6-
state, the PON is constant for at least 30-40 minutes, depending on specific experimental conditions.
40
For chronoamperometric tests, a stock solution of 250 µM SIN-1 in deoxygenated PBS buffer (pH 7.4)
was prepared and stored in leak-tight sealed vials. For cyclic voltammetric experiments, the stock
solution concentration was 1 mM SIN-1 in deoxygenated PBS buffer (pH 7.4). A solution of SIN-1 (1
-
mM) releases peroxynitrite at a rate of 1 µM min . The ONOO concentration was assessed by
1
-
-1
-1
UV/Vis measurements at λ = 302 nm for ε302 = 1705 mol cm during, as well as after, every
electroanalytical experiment, by adding a known aliquot of the stock solution to air-equilibrated PBS
41
.
The peak concentration linearly correlated with SIN-1 concentration, amounting to 1.2-3.6 % of
added SIN-1. In between experiments, solutions were typically kept on ice to minimize any
spontaneous degradation.
2.6. Electrode preparation
Glassy carbon electrodes (GCEs) were polished with alumina and diamond paste and then sonicated in
a mixture of ethanol/acetone for 30 seconds before modification by drop casting 20 µL of rGO/CoPc-
o
COOH two times (0.5 mg/mL in DMF), followed by drying in an oven at 60 C for 30 min. after each
deposition.
2.7. Instrumentation
2.7.1.X-ray photoelectron spectroscopy
X-ray photoelectron spectroscopy (XPS) experiments were performed in a PHl 5000 VersaProbe -
-9
Scanning ESCA Microprobe (ULVAC-PHI, Japan/USA) instrument at a base pressure below 5×10
mbar. Monochromatic AlK radiation was used and the X-ray beam, focused to a diameter of 100 µm,
α
was scanned on a 250×250 µm surface, at an operating power of 25 W (15 kV). Photoelectron survey
spectra were acquired using a hemispherical analyzer at pass energy of 117.4 eV with a 0.4 eV energy
step. Core-level spectra were acquired at pass energy of 23.5 eV with a 0.1 eV energy step. All spectra
were acquired at 90° between X-ray source and analyzer and with the use of low energy electrons and
low energy argon ions for charge neutralization. After subtraction of the Shirley-type background, the
core-level spectra were decomposed into their components with mixed Gaussian-Lorentzian (30:70)
shape lines using the CasaXPS software. Quantification calculations were performed using sensitivity
factors supplied by PHI.
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2.7.2.FTIR spectroscopy
Fourier transform infrared (FTIR) spectra were recorded using a ThermoScientific FTIR instrument
-
1
(
Nicolet 8700) at 4 cm . Dried rGO/CoPc-COOH (1 mg) was mixed with KBr powder (100 mg) in an
agate mortar. The mixture was pressed into a pellet under 10 tons load for 2–4 min, and the spectrum
was recorded immediately. Sixteen accumulative scans were collected. The signal from a pure KBr
pellet was substracted as the background.
2.7.3.UV/Vis measurements
Absorption spectra were recorded using a Jasco V-570 UV/VIS/NIR Spectrophotometer from Jasco
Int. Co. Ltd., Tokyo, Japan in the 200-800 nm range.
2.7.4.Raman spectroscopy
Micro-Raman spectroscopy measurements were performed at room temperature with a Horiba Jobin
Yvon LabRam HR800 spectrometer (λ=633nm). The scattered light is collected by the same objective
in backscattering configuration and detected by a CCD.
2.7.5. Electron microscopy
Scanning electron microscopy (SEM) images were obtained using an FEI Nova NanoSEM 450
scanning electron microscope equipped with a FEG (field emission gun, Schottky type) system. The
transmission electron microscopy (TEM) images were acquired using a JEOL JEM-2100 (JEOL,
Japan) transmission electron microscope operating at an acceleration voltage of 200 kV. The samples
were drop-coated from ethnolic dispersion of rGO/CPc-COOH onto carbon-coated copper TEM grids
and the solvent was evaporated under gentle heating by a UV lamp.
2.7.6.Electrochemical measurements
Cyclic voltammetry (CV), chronoamperometric (CA) and differential pulse voltammetry (DPV)
experiments were performed using an Autolab PGSTAT 101 potentiostat (Eco Chemie, Utrecht, The
Netherlands). The electrochemical cell consisted of a working electrode (GCE), Ag/AgCl as reference
electrode, and platinum wire as counter electrode. DPV experiments were performed under the
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following conditions: step potential 5 mV, modulation amplitude 25 mV, and scanning range from
0.35 V to 1.5 V.
3. Results and discussion
3.1. Caracterization of the cobalt phthalocyanine tetracarboxylic acid (CoPc-COOH) (1)
Despite the many potential applications that graphene promises to offer, one of the major challenges
4
2
remains the development of controlled functionalization schemes. Non-covalent functionalization
approaches, taking advantage of π- π stacking interactions between aromatic molecules and the
graphene basal plane, have shown to be easy and efficient means for the integration of organic
4
3-48
43
44, 45
molecules.
aminophenylbenzoic acid , hemin or tyrosine allowed simultaneous reduction of graphene oxide
GO) to reduced graphene oxide (rGO) and insertion of the organic molecules via π-π stacking
Electron donating organic molecules such as dopamine, tetrathiafulvalene,
4-
4
9
46
50
(
interactions. A comparable approach was used in this work to decorate rGO with cobalt
phthalocyanine tetracarboxylic acid (CoPc-COOH) (1) (Figure 1A). It is based on the
intercalation/adsorption of CoPc-COOH into/onto layers of reduced graphene oxide through the π-
stacking system of the two components. In the case of the phthalocyanine CoPc-COOH (1) a mixture
of four possible constitutional isomers is obtained. This mixture of isomers can be used as such in
obtaining sensing materials due to an enhanced solubility in common solvents and aqueous media
caused by the disrupting effect on the crystal lattice.
3.1.1. Electrochemistry.
0
The DMF soluble CoPc-COOH complex shows a reversible redox couple at E = -0.22 V vs. Ag/AgCl,
II
attributed to Co /Co (Figure 1B). Additional irreversible anodic bands at E = 0.65 V attributed to
I
III
II
Co /Co and E = 0.80 V corresponding to the oxidation of the phthalocyanide macrocycle are seen.
8,
22, 51
An additional band at E = -0.80 V vs. Ag/AgCl was observed under air, being significantly
decreased under argon. This band is believed to be to the one-electron catalytic oxidation of oxygen to
°-
O
2
in aprotic solvents, catalyzed by the CoPc-COOH ligand.
The possibility of this electron rich macrocycle to reduce GO to rGO and along with its
intercalation/absorption into/onto layers of rGO was achieved through the reaction of GO with CoPc-
COOH (1) at elevated temperatures (Figure 1A).
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3.1.2. XPS characterization.
The XPS analysis was performed to confirm the integration of CoPc-COOH (1) into GO and the
chemical state of GO. The presence of N1s and Co2p next to C1s and O1s evidences the presence of
CoPc-COOH (1) (Table 1). A 1/2 ratio of rGO/CoPc-COOH resulted in the highest amount of
integrated complex and was used in the following for analysis. The ratio of N/Co is ≈ 8 suggesting that
the complex is not destroyed during the chemical process. The high resolution Co2p XPS spectrum
2
Figure 2A) shows bands at 780.6, 782.2, 784.2 and 788.8 eV assigned to the Co2p3/2 band of Co ,
+
(
5
while the bands at 795.2 and 797 eV correspond to the Co2p1/2 bands. The high resolution C1s XPS
2
spectrum (Figure 2B) of GO can be deconvoluted into four peaks with binding energies at 283.7,
2
84.6, 286.6 and 287.9 eV assigned to sp -hybridized carbon, C-H/C-C, C-O and C=O species,
2
respectively. After reaction of GO with CoPc-COOH (1) with a ratio of 1/2 the XPS signature changes
2
significantly. The C1s core level spectrum shows next to the band at 284.1 eV (Csp ), contributions at
285.3 (C-C/C-H), 286.6 (C-O, C-N), 287.9 (C=O) and a small contribution at 290.1 eV (O-C=O). The
as-prepared hybrid material was further characterized using various techniques such as FTIR, Raman
spectroscopy, UV-vis, and SEM and to investigate its structure and chemical composition.
3.1.3. FTIR spectroscopy
FTIR was further employed to evidence the GO reduction in rGO/CoPc-COOH hybrid sample. Figure
2C corresponds to the transmission FTIR spectrum of GO. It comprises a broad and strong band at
-
1
∼
3400 cm assigned to the vibration of hydroxyl groups and/or adsorbed water molecules and features
due to C=O (-COOH) vibration, OH deformation, and C-O (alkoxy) and C-O (epoxy) stretching
-1
-1
modes at 1735, 1420, 1223 and 1081 cm , respectively. A band at ∼1625 cm assigned to C=C
stretching modes is also present in the FTIR spectrum of the initial GO. After reaction of GO with
CoPc-COOH forming rGO/CoPc-COOH, the intensity of the bands associated with oxygen
-1
functionalities decreased significantly. The FTIR spectrum shows a band at ∼1640 cm due to C=C
stretching modes, suggesting that the aromatic network has been restored upon reaction with CoPc-
-1
COOH. However, the persistence of O-H stretching vibration band (∼3430 cm ) as well as other
oxygen characteristic groups implies the incomplete removal of oxygen groups after GO reduction, in
line with XPS results.The C=O stretching bands of CoPc-COOH and/or residual carbonyl groups in
-1
-1
rGO are observed at 1728 cm . The red shift of C=O stretching observed in GO (1735 cm ) to 1728
-1
cm in rGO/CoPc-COOH might indicate a gain of the electron density on those groups due to electron
2
2
transfer.
1
0

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3.1.4. UV/Vis analysis
The UV-Vis absorption spectra of hydrazine-reduced rGO, CoPc-COOH (1) and rGO/CoPc-COOH in
DMF are displayed in Figure 3A. The hydrazine-reduced rGO shows an absorption band at 273 nm
2
consistent with the restoration of the sp structure in rGO. The UV/Vis spectrum of rGO/CoPc-COOH
displays similar bands as CoPc-COOH with increased absorption intensity. Compared to CoPc-
COOH, the rGO/CoPc-COOH has the Q-bands and Soret band red shifted, from 670 nm to 679 nm
and from 334 nm to 345 nm, respectively. This appears to indicate an adsorption and intercalation of
CoPc-COOH onto and into the rGO sheets. In addition, it reveals a strong π–π interaction of the CoPc-
COOH with the rGO nanosheets. For the rGO, the relocation of electrons from the graphene domain to
the CoPc-COOH causes a slightly blue shift of the absorption band of rGO from 273 nm to 268 nm,
through the π-π interaction between rGO and CoPc-COOH. The two peaks observed in the Q band
zones (609-612) nm and (670-679) nm are related to the aggregates and monomeric species,
respectively In rGo/CoPc-COOH, Q band becomes more symmetric, sharper and intense, while the
607 nm CoPc-COOH spectrum shoulder shapes into a distinct absorption band, bathochromically
shifted to 612 nm. In addition, for rGO/CoPc-COOH, both bands related to aggregates and monomer
species become intense and are red shifted, confirming that this behavior is exclusively due to
2
interactions established between rGO and CoPc-COOH and that it is not the result of the monomer-
2
5
2
dimer equilibrium.
3.1.5.Raman.
Raman spectroscopy was in addition employed and the spectrum of rGO/CoPc-COOH as it is known
as a useful technique to study the ordered/disordered crystal structures of carbonaceous materials, such
5
3, 54
as graphene nanosheets.
Figure 3B shows typical features of rGO with a broad D band at 1323
-1
-1
cm and a G band at 1576 cm , which are usually assigned to the local defects/disorders (particularly
2
located at the edges of the graphitic and graphene platelets) and the sp graphitized structures,
5
respectively. No obvious peaks corresponding to CoPc-COOH has been observed which is due to the
5
2
2
relatively- low concentration of CoPc-cOOH and the insensitivity of the Raman method. The I
D
/I
G
intensity ratio for rGO/CoPc-COOH were found to be 0.89 (initial GO I /I =1.19) with smaller I /I
D
G
D
G
peak intensity ratios corresponding to lower defect/disorders in a graphitized structure such a rGO and
2
the improvement in the sp graphitized rGO/CoPc-COOH structure.
1
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3.1.6 Electron microscopy.
The TEM images revel that rGO/CoPc-COOH consist of randomly aggregated, crumpled sheets
Figure 3C). The surface morphology of the rGO/CoPc-COOPH matrix was done by SEM and
showes that rGO/CoPc-COOH has a curled, nonporous structure (Figure 3D) as observed for other
(
5
6
rGO/cobalt nanocomposites.
3.2. Electrocatalytic response rGO/CoPc-COOH modified glassy carbon electrodes
The electrochemical behavior of rGO/CoPc-COOH modified GCE in DMF is depicted in Figure 4A.
III
II
An irreversible redox band at 0.72 V vs. Ag/AgCl attributed to the Co /Co redox couple is observed.
Two redox bands at -0.31 V vs. Ag/AgCl and -0.53 V vs. Ag/AgCl are seen on the cathodic scan.
Zagal and co-workers observed a similar behavior on carbon electrodes modified with a commercially
3
available cobalt phthalocyanine. The first band was attributed to multilayer stacking of cobalt
II
phthalocyanine, while the band at more negative potential was attributed to the Co /Co redox couple.
I
3.2.1. Detection of peroxynitrite
The electrocatalytic behavior of rGO/CoPc-COOH modified glassy carbon electrodes towards the
5
7
oxidation of peroxynitrite was investigated. Though several strategies based on biochemical assays
5
8-62
63
or electron (paramagnetic) spin resonance (ESR) spectroscopy have
optical via suitable dyes
-
been developed for peroxynitrite detection, electrochemical quantification of ONOO has come into
4
,
the forefront when it comes to real-time, label-free and direct measurements of these reactive species.
19, 20, 64-66
Different strategies to synthesize PON were published and have been recently reviewed in some
6
4
14, 18
detail. While sufficiently stable for 30 to 60 minutes in alkaline solutions (pH ≈10 ),
6
peroxynitrite decomposes in less than 1 s at neutral pH. Since using alkaline buffer PON solutions is
7
6
8, 69
customary in electrochemical sensing methods,
the following experiments were conducted in
CAPS buffer (pH 10). Figure 4B shows the performance of the rGO/CoPc-COOH modified GCE in
the presence of peroxynitrite. An increase in the oxidation current observed from 0.72 V vs. Ag/AgCl
onwards is attributed to the electrocatalytic oxidation of peroxynitrite:
ꢂꢂ
ꢂꢂꢂ
ꢈ
ꢀ
ꢁ ꢃꢄ − ꢀꢅꢅꢆ → ꢀꢁ ꢃꢄ − ꢀꢅꢅꢆ + ꢇ
ꢂꢂꢂ
ꢈ
ꢂꢂ
°
ꢀ
ꢁ ꢃꢄ − ꢀꢅꢅꢆ + ꢅ = ꢉ − ꢅ − ꢅ → ꢀꢁ ꢃꢄ − ꢀꢅꢅꢆ − ꢅꢅꢉꢅ
1
2

Page 13 of 27
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DOI: 10.1039/C4RA09781E
ꢂꢂ
°
ꢈ
ꢂꢂꢂ
°
ꢀ
ꢁ ꢃꢄ − ꢀꢅꢅꢆ − ꢅꢅꢉꢅ + ꢇ → ꢀꢁ ꢃꢄ − ꢀꢅꢅꢆ + ꢅꢉꢅ
ꢊ
°
2
ꢅꢉꢅ → ꢅ + ꢉꢅ
ꢊ
ꢊ ꢊ
While synthetic peroxynitrite alkaline buffer solutions help the general sensor development, as one
comes closer to test peroxynitrite in vitro and in vivo, working under physiological pH is primordial.
Consequently, the use of the PON donor 3-morpholino-sydnonimine (SIN-1) is gaining ground,
7
0, 71
especially for optical sensing techniques.
Using peroxynitrite formed from SIN-1, rGO/CoPc-
COOH modified GC electrode showed a comparable electrocatalytic behavior at pH 7.4 to that
obtained in alkaline media (Figure 4B). This strongly indicates that the rGO/CoPc-COOH modified
GC electrode could potentially be used for in vivo conditions.
The sensitivity of the sensor was determined using chronoamperometry (Figure 5A). The calibration
-
curve in Figure 5B indicates that the oxidation current scales linearly with increasing ONNO
-1
concentration. The sensitivity towards peroxynitrite was ≈11.5±1.0 nA nM with a limit of detection
of ≈1.7 nM and a linear range up to 20 nM of peroxynitrite. The detection limit is an order of
magnitude lower than that on manganese tetraaminophthalocyanine modified platinum or Pt/C
1
microelectrodes (LOD = 5 µM), but is also inferior than reported on nanostructured polymerized
4
18
EDOT/hemin carbon fiber microelectrodes (LOD=200 nM) , poly(cyanocobalamin)-modified GE
7
2
20
(
LOD=100 nM) and GC/rGO/hemin (≈5 nM).
The selectivity of the modified electrode was evaluated in the presence of various interfering
substances. To account for higher concentrations of interfering species present in biological
samples, the interference of a 1000 fold excess of the interfering analytes over the
peroxynitrite species was examined (Figure 6A), namely nitrite, nitrate, hydrogen peroxide,
dopamine, ascorbic acid and glucose. The results indicated that the rGO/CoPc-COOH
modified GC electrode has, beside nitrite, interference-free signals for peroxynitrite
measurements similar to in vivo conditions. The electrocatalytic oxidation of nitrite on
3, 73, 74
modified carbon based electrodes is reported to occur between 0.8-1.1 V/SCE.
Figure
6B shows the differential pulse voltammogram of nitrite, peroxynitrite and when in a mixture
with nitrite 12 times more concentrated. The rGO/CoPc-COOH modified GC electrode shows
two distinguished redox potential for nitrite and peroxynitrite, being most possible the reason
for the good selectivity.
The reproducibility of the rGO/CoPc-COOH modified GC electrode was investigated by
measuring the current signal for 10 nM PON at five modified electrodes prepared under the same
experimental conditions. A relative standard deviation (RSD) of 5.6 % was determined, indicating a
good reproducibility of the fabrication method. The long-term stability of the electrode was examined
1
3

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after storage in a refrigerator at 4°C for a 3 week period. The sensor retained about 95.6% of its initial
current response to 10 nM PON. The results demonstrate that the sensor exhibits a good stability.
3.1.2. Detection of hydrogen peroxide
Figure 7A shows a typical cyclic voltammogram of the rGO/CoPc-COOH modified electrode in 0.1
-1
M PBS in the absence of H
electrochemical response. Upon addition of 2 mM H
observed, indicating a good electrocatalytic activity of the interface towards hydrogen peroxide
reduction. The electrocatalytic responses of the electrode towards H was further investigated by
amperometric current-time response upon successive addition of different concentrations of H
Figure 7B). The reduction current increases gradually upon injection of increasing concentrators of
into the PBS solution. From the corresponding calibration curve (Figure 7C), a linear
relationship between current and H concentration from 0.1-12 mM could be identified with an
2
O
2
at a scan rate of 50 mV s . The modified electrode exhibits no obvious
2
O
2
, a remarkable increase of the current was
2
O
2
2
O
2
(
2 2
H O
2
O
2
-1
estimated sensitivity of 14.5 µA mM . A detection limit of ≈60 µM at a signal-to-noise ratio of 3 was
achieved using the rGO/CoPc-COOH modified electrode. The detection limit for the sensors is in the
7
5-77
same order of magnitude (1-10 µM) than other hydrogen peroxide sensor.
was comparable to the PON case. The sensor retained 96.9% of its initial current response to 5 mM
when stored in a refrigerator at 4°C for 3 week time. The proposed strategy for the preparation of
The long-term stability
2 2
H O
the matrix is moreover straightforward and simple. It furthermore allows the parallel detection of
peroxynitrite without any interference.
4. Conclusion
We have demonstrated that the reaction of GO and cobalt phthalocyanine tetracarboxylic acid (CoPc-
COOH) (1) allows for the simultaneous reduction/functionalization of rGO with (1). The resulting
rGO/CoPc-COOH matrices were successfully applied for the determination of peroxynitrite and
hydrogen peroxide. Compared with other electrochemical method for the detection of peroxynitrite,
the rGO/CoPc-COOH modified GC electrode reported here has several advantages such a nanomolar
sensitivity, high selectivity, good stability and response time of about 15 s. These results open an
interesting perspective for the use of such electrodes to detect peroxynitrite in real biologic samples.
Miniaturization of the sensor might be an important step for achieving this goal. It is known that ultra-
small sensors can afford rapid responses and better analyte sensitivity along with high spatial
1
4

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resolution and use in non-stirred solutions or soft solid matrices. Ongoing work aims to miniaturize
these peroxynitrite and hydrogen peroxide sensitive-selective electrocatalytic interfaces.
Acknowledgements
Q. W. thanks the “Fundamental Research Funds of Shandong University” under grant N°
31370074614039. R.B. and S.S. gratefully acknowledge financial support from the Centre National de
Recherche Scientifique (CNRS), the University Lille 1 and Nord Pas de Calais region. S.S thanks the
Institut Universitaire de France (IUF) for financial support. A.V. was supported by the Autoritatea
Nationala Romana pentru Cercetare Stiintifica (CNDI - UEFISCDI) through project PN-II-PT-PCCA-
2011-3.1-1809 and V.R. was funded by CNDI-UEFISCDI through the project 3.2-1391. S.F.P. and
I.S.H. acknowledge funding by CNDI-UEFISCDI, project PN-II PCE-2011-3-1076 and by Institut
Français de Roumanie for Partenariat BHC 28684VM. Support from the European Union through
FP7-PEOPLE-IRSES (No. 269009) is also acknowledged. Raluca Oprea, Ana-Maria Popescu and
Veronica Andrei are credited for their initial assistance during the preliminary phase of this work.
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Table1: Composition in atomic percentage of the resulting rGO/CoPC-COOH as a function of the
initial concentration ratio of GO/CoPC-COOH.
Ratio GO/CoPC-COOH C1s
O1s
33.8
32.2
22.5
25.3
27.7
N1s
-
Co2p
-
C/O
1.96
1.97
2.10
2.35
2.12
GO only
66.2
63.5
45.1
59.4
58.7
1
1
1
1
/0.5
3.8
0.5
3.6
1.7
1.5
/1
28.8
13.6
12.1
/2
/4
2
0

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(
A)
(
B)
50
III
Co / Co
II
II
Co / Co
I
0
°-
O /O2
2
-50
-
1
-0,5
0
0,5
1
E / V vs. Ag/AgCl
Figure 1: (A) Synthesis of rGO/CoPc-COOH, (B) cyclic voltammogram of CoPc-COOH (1, 10 mM)
-1
4
in DMF/0.1M TBATBF , scan rate= 100 mV s under argon (black), in air (blue).
2
1

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(
A)
(B)
1
1
1
1
1
1
1
300
250
200
150
100
050
000
rG O / C oP h-C O O H (1)
2
1. sp C
2
p3/2
Co2p
1
2
3
4
. C -C /C -H
. C -O /C -N
. C = O
2p1/2
5. C O O H
2
2
4
3
5
(
b )
3
G O
2
1
. sp
C
2. C -C /C -H
. C -O
4. C = O
3
9
9
50
00
4
(
a)
280
1
2
94
292
290
288
286
284
282
8
08 804 800 796 792 788 784 780 776
B inding energy / e V
binding energy / eV
(
C)
rGO/CoPC-COOH
GO
4000 3500 3000 2500 2000 1500 1000
-1
Wavenumber / cm
Figure 2: High resolution XPS spectra of (A) Co2p and (B) C1s of GO (a) and rGO/CoPc-COOH (b);
C) FTIR spectra of GO (black) and rGO/CoPc-COOH (green)
(
2
2

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(
A)
(B)
2
500
G
rGO/CoPc-COOH
CoPc-COOH
rGO
rGO/CoPc-COOH
0
0
0
0
,4
,3
,2
,1
0
D
2000
1
1
500
000
500
2
D
3
20 400 480 560 640 720 800
1000
1500
2000
2500
3000
-1
wavelength / nm
Wavenumber/cm
(
C)
(D)
Figure 3: (A) UV/Vis spectra of CoPc-COOH (1 mM) in DMF (black line), hydrazine reduced GO (1
mg /mL) in DMF (blue ) and of rGO/CoPc-COOH (1 mg/mL) in DMF (green ); (B) Raman Spectrum
of rGO/CoPc-COOH ; (C) TEM of rGO/CoPc-COOH, (D) SEM image of rGO/CoPc-COOH.
23

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DOI: 10.1039/C4RA09781E
(
A)
1
0
5
0
5
III
Co / Co
II
-
-
10
15
II
Co / Co
I
-
-
1
-0,5
0
0,5
1
E / V vs. Ag/AgCl
(
B)
200
150
100
pH = 7.4
pH = 10
50
0
-
50
-
1
-0,5
0
0,5
1
E / V vs. Ag/AgCl
Figure 4: Cyclic voltammograms recorded on GCE modified by drop-casting rGO/CoPc-COOH in
CAPS buffer (pH 10) in (A) the absence and in (B) the presence of 15 nM peroxynitrite in CAPS
-1
buffer (pH 10, blue) or PBS (pH=7.4) 15 nM (black), scan rate: 100 mV s .
2
4

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DOI: 10.1039/C4RA09781E
(
A)
(B)
500
400
300
200
100
0
3
3
2
2
1
1
50
00
50
00
50
00
50
0
0
20
40
60
80
100
0
5
10
15
20
time / min
[peroxynitrite] / nM
Figure 5. (A) Amperometric response curve in PBS (pH=7.4) obtained using glassy carbon electrode
modified with rGO/CoPc-COOH by drop-casting polarized at 1.1 V vs. Ag/AgCl with subsequent
addition of petoxynitrite, (B) Calibration curve
2
5

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(
A)
200
150
100
400
350
300
250
200
150
100
50
0
4
8
12
16
20
t/min
50
0
-
PON NO2
-
H O
2 2
AA
Gl
NO3
DA
(
B)
3
00
50
00
50
00
1
1
1
5 nM PON
80 µM nitrite
80 µM nitrite + 15 nM PON
PON
2
2
1
1
nitrite
50
0
0
,5 0,6 0,7 0,8 0,9
1
1,1 1,2
E / V vs. Ag/AgCl
Figure 6: Comparative study of the oxidative current determined on a set of rGO/CoPc-COOH
modified CGEs for peroxynitrite (15 nM) and for interfering species: nitrate, nitrite, hydrogen
peroxide, dopamine (DA), ascorbic acid (AA), glucose (Gl) at 1000 times excess (15 µM), (B) DPV of
nitrite (180 µM, blue), peroxynitrite (15 nM, black) and their mixture (red).
2
6

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(
A)
(B)
2
1
0
0
0
-50
-40
-30
-20
-10
0
-
-
-
-
10
20
30
40
(
a)
(
b)
-0,4
-
1
-0,8
-0,6
-0,2
0
0,2
1
0
15
20
25
30
35
40
E vs. (Ag/AgCl) / V
t / min
(
C)
-
100
-
-
-
-
80
60
40
20
0
-
-
-
-
3,5
-
3
2,5
-
2
1,5
-
1
0,5
0
0
100
200
300
400
500
600
H O2 / µM
2
0
5
10
15
20
25
30
35
H O / mM
2
2
Figure 7: (A) Cyclic voltammograms of rCO/CoPc-COOH modified CGE in N
2
saturated 0.1 M PBS
-1
solution (pH 7.4) in the absence (grey, a) and presence (black, b) of 2 mM H O , scan rate=50 mV s ;
2
2
(
B) Amperometric response of rCO/CoPc-COOH modified CGE polarized at -0.50 V vs. Ag/AgCl in
-saturated 0.1 M KCl with subsequent addition of H (500 µM), (C) Calibration curve of
rCO/CoPc-COOH modified CGE polarized at -0.50 V vs. Ag/AgCl in N -saturated 0.1 M KCl
N
2
2 2
O
2
2
7