A non-covalent interaction of Schiff base copper alanine complex with green synthesized reduced graphene oxide for highly selective electrochemical detection of nitrite

Article abstract of DOI:10.1039/C6RA20580A

A novel and selective nitrite sensor based on non-covalent interaction of Schiff base copper complex [Cu(sal-ala)(phen)] with reduced graphene oxide (RGO) was developed by simple eco-friendly approach. The morphology of the prepared RGO/[Cu(sal-ala)(phen)] nanocomposite was characterized by scanning electron microscopy (SEM), ultraviolet visible spectroscopy (UV), electrochemical impedance spectroscopy (EIS), energy dispersive X-ray spectroscopy, X-ray diffraction studies and Fourier transform infrared spectroscopy (FT-IR). On the other hand, the electrochemical studies of the prepared nanocomposite was investigated by cyclic voltammetry (CV) and amperometric technique. The RGO/[Cu(sal-ala)(phen)] nanocomposite modified glassy carbon electrode (GCE) exhibit the higher electrocatalytic activity towards detection of nitrite. Moreover, the RGO/[Cu(sal-ala)(phen)] modified GCE was determined the nitrite with low detection limit (19 nM), broad linear range (0.05-1000 μM) and high sensitivity (3.86 μA μM^?1 cm^?2). Besides, the proposed sensor shows good selectivity, repeatability, reproducibility and long term operational stability. The appreciable recoveries was achieved for the detection of nitrite in water and sausage samples, which imply the practical feasibility of the modified electrode.

Full text of DOI:10.1039/C6RA20580A

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Adv., 2016, DOI: 10.1039/C6RA20580A.
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Page 1 of 26
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DOI: 10.1039/C6RA20580A
A nonꢀcovalent interaction of Schiff base copper alanine complex with green
synthesized reduced graphene oxide for highly selective electrochemical
detection of nitrite
a
a
ac
Subramanian Sakthinathan , Subbiramaniyan Kubendhiran , ShenꢀMing Chen* , Fahad M.A.
c
a
b
*b
c
c
AlꢀHemaid , Wei Cheng Liao , P.Tamizhdurai , S. Sivashankar , M. AjmalAli , A.A. Hatamleh
a
Electroanalysis and Bioelectrochemistry Lab, Department of Chemical Engineering and
Biotechnology, National Taipei University of Technology, No.1, Section 3, ChungꢀHsiao East
Road, Taipei 106, Taiwan (R.O.C).
b
National Centre for Catalysis Research, Indian Institute of Technology, Chennaiꢀ600036, India.
c
Department of Botany and Microbiology, College of Science, King Saud University, Riyadh
11451, Saudi Arabia
*
Corresponding Author
a
(S.M. Chen). Fax: +886 2270 25238; Tel: +886 2270 17147
Eꢀmail address: smchen78@ms15.hinet.net
b
(S. Sivashankar) NCCR, IIT, Chennai.
Eꢀmail address: ssivasanker@iitm.ac.in
1

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Abstract
A novel and selective nitrite sensor based on nonꢀcovalent interaction of Schiff base copper
complex [Cu(salꢀala)(phen)] with reduced graphene oxide (RGO) was developed by simple ecoꢀ
friendly approach. The morphology of the prepared RGO/[Cu(salꢀala)(phen)] nanocomposite
was characterized by scanning electron microscopy (SEM), ultraviolet visible spectroscopy
(UV), electro chemical impedance spectroscopy (EIS), energy dispersive xꢀray spectroscopy, xꢀ
ray diffraction studies and Fourier transform infrared spectroscopy (FTꢀIR). On the other hand,
the electrochemical studies of the prepared nanocomposite was investigated by cyclic
voltammetry (CV) and amperometric technique. The RGO/[Cu(salꢀala)(phen)] nanocomposite
modified glassy carbon electrode (GCE) exhibit the higher electrocatalytic activity towards
detection of nitrite. Moreover, the RGO/[Cu(salꢀala)(phen)] modified GCE was determined the
nitrite with low detection limit (19 nM), broad linear range (0.05ꢀ1000 µM) and high sensitivity
ꢀ2
(
3.86 µA/µM cm ). Besides, the proposed sensor shows a good selectivity, repeatability, reprod
ucibility and long term operational stability. The appreciable recoveries was achieved for the
detection of nitrite in water and sausage samples, which implying that the practical feasibility of
the modified electrode.
Keywords
Reduced graphene oxide, Schiff base copper complex, Nitrite, Green synthesis, Oxidation,
Electrocatalysis, Sensitivity, Selectivity.
2

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1. Introduction
Nitrites is an inorganic compound and it is widely used in food additives, corrosion
1
ꢀ2
inhibitors and fertilizing agents. It can easily contaminated the drinking water due to the
3
human activities, industrial pollutants and fertilizing agents. The maximum permissible level of
nitrite in drinking water is 1 mg/L and the excess of nitrite can causes several health problems
like blue baby syndrome and shortness of breath. Moreover, nitrite can readily react with
4
,5
hemoglobin to form methemoglobin, which makes the oxygen deficiency in blood. Besides,
nitrite can react with various amines to the formation of Nꢀnitrosamines and it causes stomach
cancer to human. Therefore, it is very important to determination of nitrite in food, drinking
6
water and environmental samples. Nowadays, several analytical methods have been developed
7
8
for the detection of nitrite including, chromatographic methods , capillary electrophoresis,
9
10
11
chemiluminescence , ion exchange chromatographic and electrochemical methods. Compared
with other traditional analytical methods, electrochemical methods are widely used for the
1
2,13
detection of nitrite due to the high sensitivity, simplicity and selectivity.
The unmodified
electrodes are active towards detection of nitrite. However, the working potential was higher and
selectivity also poor in the presence of interfering compounds. Hence, the chemically modified
electrodes were used for the detection of nitrite due to the lowest oxidation potential and higher
sensitivity.
2
Graphene is a two dimensional hexagonal carbon lattice with sp hybridization. It has a wide
spread applications because of its unique physicochemical properties such as high surface area,
1
4,15
chemical stability, high electrical conductivity and extraordinary mechanical properties.
Comparatively, RGO has superior conductivity than GO due to the more active edge plane
1
6
defects and large effective surface area, so we need to find a suitable methods for reducing GO.
Nowadays, different kinds of methods have been used for the preparation of RGO, which
17
18
19
includes chemical vapor deposition (CVD), mechanical exfoliation, electric arc discharge,
2
0
21
thermal reduction and chemical reduction. Among all methods, the chemical reduction is
more suitable and versatile method for the preparation of RGO in bulk quantities. However, in
this method toxic and explosive chemicals are used for the RGO preparation. Therefore, we need
2
2
to explore environmentally friendly reducing agents for effective reduction of GO. Incidentally,
the caffeic acid provides the good platform to the reduction of GO, because the caffeic acid (CA)
3

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is one of the hydroxyl cinnamic acids and it has well antioxidant properties. Moreover, the
structure of CA has two adjacent hydroxyl groups an aromatic ring, which helps donation of
hydrogen to the GO reduction. Therefore, CA considered as a green, effective and low cost
2
3
reducing agent for the RGO preparation.
Schiff base complexes have an azomethine group (−HC=N−) that plays an important role in coꢀ
ordination chemistry. The Schiff bases are used to form stable complexes with transition metal
ions. In addition, the Schiff base complexes are functionalized with RGO and used for different
24ꢀ26
kinds of applications like sensors, catalysis and energy storage applications.
Besides, the
nonꢀcovalent functionalization has advantages than covalent functionalization, because it avoids
the destruction and retaining the unique properties of graphene. Moreover, the nonꢀcovalent
2
7, 28
interaction was enhanced the mechanical and electrical properties of the nanocomposite.
this work we have synthesized the RGO/[Cu(salꢀala)phen] (salꢀala = salicylalanineꢀalanine, phen
1, 10– phenanthroline) nanocomposite by nonꢀcovalent interaction. To the best of our
In
=
knowledge for the first time Schiff base copper complex [Cu(sal–ala)phen] noncovalent
interaction with RGO to prepare a new type of nanocomposite for the electrochemical detection
of nitrite.
2. Experimental
2.1 Reagent and solution
Graphite powder (size ˂ 20 µm), sodium nitrite, caffeic acid, Lꢀalanine were purchased from
sigma aldrich. 1, 10ꢀphenanthroline, copper (II) chloride monohydrate and salicylaldehyde were
purchased from Merck chemical. The phosphate (0.05 M) buffer solution (PBS) was prepared by
using Na HPO and NaH PO , the pH was adjusted by H SO or NaOH. All the chemicals used
2
4
2
4
2
4
in this work were of analytical grade and used without any further purification. Prior to each
experiment, the electrolyte solutions were deoxygenated with purified N for 15 min. All the
2
experiment was carried out at room temperature and all the required solutions were made up by
double distilled water.
2.2 Experimental apparatus
4

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Electrochemical studies were carried out at the CHI 410 electrochemical work station. The
electrochemical cell contains GCE as a working electrode, Ag/AgCl as a reference electrode and
Pt wire as a counter electrode. The electrochemical experiment was performed at N saturated
2
electrolyte. UVꢀvisible spectra were taken by Perkinelmer and electrochemical impedance
spectra were carried out by the EIM6EX ZAHNER, Kroanch, Germany. Scanning electron
microscope studies were carried out using a Hitachi Sꢀ3000H microscope. Elemental analysis
was carried out using HORIBA EMAX XꢀACT (model 51ꢀADD0009). Powder Xꢀray diffraction
(
XRD) studies were performed XPERTꢀPRO (Netherlands) diffractometer using Cu Kα radiation
k=1.54 Å). Fourier transform infrared spectroscopy (FTꢀIR) was carried out using the Jasco FTꢀ
(
IR 6600 spectrometer.
.3 Preparation of [Cu (salꢀala)(phen)] complex
The inorganic complex was synthesized based on previous report with slight modification.
2
2
9ꢀ32
1
,10ꢀphenanthroline (5 mmol; 0.99 g in 20 mL methanol) and salicylalanine (5 mmol; 0.97 g in
0 mL methanol) ligands were mixed and homogenized. Then the homogenized solution was
2
added dropwise to a solution of CuCl .H O (5 mmol; 0.86 g in 20 mL water) while stirring at 50
2
2
°
C. The bright green precipitate of [Cu(salꢀala)(phen)] complex was obtained (scheme 1), this
precipitate was filtered and washed with ethanol then dried in a desiccator. The prepared Schiff
base complex was confirmed by the UV visible and FTꢀIR spectroscopy.
2.4 Preparation of RGO/[Cu(salꢀala)(phen)] inorganic nanocomposite
3
3
Graphene oxide was prepared from graphite by modified hummers method. The RGO was
3
4
prepared by the following procedure of Bo et al method. 10 mg of GO was dispersed in 100
mL deionized water and caffeic acid (CA) (5 mg ) was added in to the GO dispersion. The
mixture was then heated at 95° C for 24 hours in an oil bath with the assistance of stirring. The
resulting suspension was collected and washed with deionized water and ethanol. Finally, the
prepared RGO was dried at 35° C under vacuum condition.
The nonꢀcovalent approach was used to prepare the RGO/[Cu(salꢀala)(phen)] nanocomposite
3
5
with distinctive electrocatalytic activity towards the oxidation of nitrite. The nanocomposite
was synthesized by dispersing the RGO in DMF and further addition of [Cu(salꢀala)(phen)]
under vigorous stirring. Then, the obtained homogeneous solution was subjected to ultrasonic
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treatment (30 min) for enhancing the interaction between RGO and [Cu(salꢀala)(phen)]. The
resultant homogeneous RGO/[Cu(salꢀala)(phen)] nanocomposite was filtered and washed with
ethanol three times and dried in oven. Finally, the prepared RGO/[Cu(salꢀala)(phen)] was reꢀ
dispersed in DMF (1 mL) and drop casted on the cleaned GCE surface then dried in room
temperature. The scheme 2 displays the preparation of GCE/RGO/[Cu(salꢀala)(phen)] modified
electrode for the electrochemical detection of nitrite.
3. Result and discussion
3.1 characterization of prepared nanocomposite
Fig 1 shows the SEM images of (A) GO, (B) RGO, (C) GO/[Cu(salꢀala)(phen)], (C’) [Cu(salꢀ
ala)(phen)], (D) RGO/[Cu(salꢀala)(phen)]. The SEM image of graphene oxide shows the thin and
wrinkled surface. In addition, RGO exhibited randomly aggregated with distinct edges and
folding sheet surfaces. The SEM image of GO/[Cu(salꢀala)(phen)] exhibit the Schiff base copper
complex incorporated on the GO surface and the inset Fig C’ shows the SEM image of [Cu(salꢀ
ala)(phen)] complex. The SEM image of RGO/[Cu(salꢀala)(phen)] nanocomposite can be
observed that the [Cu(salꢀala)(phen)] inorganic complex was uniformly covered on the RGO
surface. Furthermore, the elemental composition of asꢀprepared nanocomposite was explored by
elemental analysis. Fig 1E and F displayed the EDX spectra of RGO/[Cu(salꢀala)(phen)]
nanocomposite. Where, the signals for carbon, oxygen, nitrogen and copper confirmed the
formation of nanocomposite. Notably, the signals of copper and nitrogen confirms the Schiff
base formation and complexation.
EIS has been used to understand the charge transfer resistance (R ) of the different modified
ct
electrodes at the electrode and electrolyte interface. Fig 2A shows the EIS plots of (a) bare GCE,
(b) GCE/RGO, (c) GCE/[Cu(salꢀala)(phen)], (d) GCE/GO/[Cu(salꢀala)(phen)], (e) GCE/RGO/
3
ꢀ/4ꢀ
[
Cu(salꢀala)(phen)] in 5 mM [Fe(CN)6]
with 0.1 M KCl as a supporting electrolyte. The
diameter of the semicircle is corresponding to the charge transfer resistance (R ) of the modified
ct
electrodes. The higher R value of about 1561 ꢁ was observed for the GCE/[Cu(salꢀala)(phen)]
ct
modified electrode. The bare GCE and GCE/RGO shows the R value of 378 ꢁ and 244 ꢁ
ct
respectively. However, the Rct value of GCE/GO/[Cu(salꢀala)(phen)] modified electrode
observed about 235 ꢁ, the charge transfer resistance of Schiff base copper complex was
6

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decreased due to the nonꢀcovalent interaction with GO. Interestingly, the GCE/RGO/[Cu(salꢀ
ala)(phen)] modified electrode shows the low R value of 33 ꢁ due to the reduction of GO to
ct
RGO, which enhance the conductivity of the modified electrode. The EIS results confirmed that
the GCE/RGO/[Cu(salꢀala)(phen)] modified electrode have higher electron transfer properties
with low charge transfer resistance. The UVꢀVisible spectra was used to identify the structural
information about the nanocomposites. Fig 2B shows the UV visible spectra of (a) RGO, (b)
[
Cu(salꢀala)(phen)] and (c) RGO/[Cu(salꢀala)(phen)]. The synthesized RGO shows the
absorbance peak at 283 nm, it confirms that the GO was successfully reduced by CA with the
ecoꢀfriendly route. Furthermore, the absorbance peak at 222 nm for the πꢀπ* transition of
phenanthroline and 272 nm for nꢀπ* transition of the salicylidene. The Cu (II) complex exhibited
the UV visible spectra at 372 nm due to the ligand to metal charge transfer transition and the
RGO/[Cu(salꢀala)(phen)] shows the peaks that observed in the free base confirms the formation
3
6
of nanocomposite.
The Fig 3A shows the Xꢀray diffraction studies of (a) RGO, (b) [Cu(salꢀala)(phen)], (c)
RGO/[Cu(salꢀala)(phen)]. The XRD pattern of RGO shows the broad peak at 26° due to the short
range order for stacked layer. On the other hand, the XRD spectra of [Cu(salꢀala)(phen)] shows
the peaks at 11º, 12.6º, 17º, 19º, 22º and 25º. Which confirms the formation of schiff base copper
(
II) complex. Besides, the XRD spectra of RGO/[Cu(salꢀala)(phen)] observed at 11°, 12°, 25°,
3
7
3
0°, 41° and 69° are quite similar to the [Cu(salꢀala)(phen)]. However, the intensity has fall
down and the peak shapes are changed. This results confirmed that the formation of
RGO/[Cu(salꢀala)(phen)] nanocomposite. The surface chemistry, bonding nature and chemical
structure of the prepared nanocomposite was characterized by FTꢀIR spectra. Fig 3B shows the
FTꢀIR spectra of (a) RGO, (b) [Cu(salꢀala)(phen)], (c) RGO/[Cu(salꢀala)(phen)]. The FTꢀIR
ꢀ1
spectrum of RGO exhibited the characteristic peak at 3452 cm for the stretching vibration of
ꢀ1
ꢀ1
hydroxyl groups. Moreover, the peaks observed at 1725 cm and 1229 cm are corresponding to
ꢀ1
the stretching vibration of C=O and CꢀO. The stretching vibration peak appeared at 833 cm for
the CꢀH rocking vibration which confirms the phenanthroline functionalities in [Cu(salꢀ
−1
ala)(phen)]. The peak in the region of 2063 cm for NꢀH bonds of Schiffꢀbase complex. The Cuꢀ
−
1
−1
O band appeared at 930 cm and CuꢀN appeared at 528– 549 cm . The peak at around 2918
−
1
−1
cm and 2892 cm can be assigned to C–H and CH stretching vibration respectively. The FTꢀ
2
IR spectra of RGO/[Cu(salꢀala)(phen)] nanocomposite shows the stretching vibration similar to
7

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3
3
the RGO and [Cu(salꢀala)(phen)]. The FTꢀIR results confirmed that the Schiff base copper
complex has been nonꢀcovalently interact with RGO and leads to the composite formation.
4. Electrocatalytic oxidation of nitrite
4.1 Electrocatalytic oxidation of nitrite at various modified electrodes and effect of
different concentration
Fig 4A shows the CV curves of (a) RGO/[Cu(salꢀala)(phen)] nanocomposite modified electrode
in the absence of nitrite and (b) bare GCE, (c) GO, (d) [Cu(salꢀala)(phen)], (e) RGO, (f)
GO/[Cu(salꢀala)(phen)], (g) RGO/[Cu(salꢀala)(phen)] nanocomposite modified electrodes in the
presence of 200 µM nitrite in PBS (pH 5). The bare GCE and GO modified electrodes oxidize
the nitrite at the peak potential (E ) of 0.99 V and 0.98 V with a corresponding oxidization peak
P
current of (I ) 25.23 µA and 27.38 µA respectively. In addition, the [Cu(salꢀala)(phen)] modified
P
electrode displays the E and I of about 1.056 V and 30.64 µA. Besides, the RGO modified
P
P
electrode exhibits the E of 0.964 V and the I of 31.33 µA for the oxidation of nitrite.
P
P
Furthermore, the GO/[Cu(salꢀala)(phen)] modified electrode shows the value of E and I at 0.93
P
P
V and 43.9 µA respectively for the oxidation of nitrite. Comparatively the oxidation peak current
of nitrite was increased for GO/[Cu(salꢀala)(phen)] modified electrode than that of [Cu(salꢀ
ala)(phen)] and RGO modified electrodes. These results confirmed that the formation of nonꢀ
covalent interaction between the GO and [Cu(salꢀala)(phen)], which enhanced the catalytic
activity. Generally, the RGO shows the better electrocatalytic properties than GO because the
GO contains many oxygen functionalities, which creates an internal resistance at the modified
electrode. In this aspect, the RGO/[Cu(salꢀala) (phen)] nanocomposite was prepared and applied
for the determination of nitrite alternative to the GO/[Cu(salꢀala)(phen)]. As expected, the
RGO/[Cu(salꢀala)(phen)] nano composite exhibits the substantial oxidization peak potential at
(
E ) 0.97 with an improved oxidation peak current (I ) of 49.80 µA, which are comparatively
P
P
higher than that of all aforementioned modified electrodes. These results can be attributed to the
nonꢀcovalent interaction of RGO and [Cu (salꢀala) (phen)], which plays an important role in the
oxidation of nitrite due to their extraordinary electro catalytic activity. Hence, the RGO/[Cu(salꢀ
ala)(phen)] nanocomposite modified electrode as an excellent electrode material for nitrite
sensor. Fig 4B shows the CV curves of RGO/[Cu(salꢀala)(phen)] nanocomposite modified
electrode for the oxidation of nitrite in various concentrations of nitrite containing PBS (pH 5) at
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ꢀ1
the scan rate 50 mVs . Increasing the concentration of nitrite from 50 µM to 600 µM, the
oxidized peak current also increased linearly. The Fig 4B inset shows the linear relation between
the peak current vs concentration and the corresponding linear regression equation can be
2
expressed as I =0.5074x ꢀ 40.813 with a correlation coefficient R =0. 9947. It is evident that
pa
the RGO/[Cu(Salꢀala)(phen)] nanocomposite modified GCE electrode has the good
electrocatalytic activity towards the detection of nitrite.
4.2 Effect of pH and scan rate
The influence of pH in the performance of an asꢀprepared modified electrode was investigated
for the detection nitrite using CV. The CV experiment was recorded at RGO/[Cu(salꢀala)(phen)]
modified GCE in various pHs ranging from 1 to 9 towards the electrocatalytic oxidation of 200
µM nitrite. As shown in the Fig 5A, the oxidation peak current of the nitrite was increased when
increasing the electrolyte pH from 1 to 5 afterwards it decreases by varying the pH 6 to 9. This
ꢀ
result stated that, at lower pH the NO ions are unstable so it can be easily converted to NO and
2
ꢀ
NO . At the same time in higher pHs, the proton deficiency was occurred. However, at pH 5 the
3
sensor exhibited higher electro catalytic performance (Fig 5B). Therefore, we have choose pH 5
for further electrochemical studies. Fig 6A shows the typical CV curve obtained at the
RGO/[Cu(salꢀala)(phen)] modified electrode for the different scan rates in PBS (pH 5)
containing 200 µM nitrite. The result showed that the peak current was linearly increased with
ꢀ1
respect to the increasing scan rates from 0.01 to 0.11 Vs . Fig 6B shows corresponding plot of
1
/2
peak current (I ) Vs square root of scan rate (ν ) and calculated the corresponding linear
p
1
/2
−1 1/2
2
regression equation as I = 75.58 ν (Vs )
+ 10.60 with R = 0.998. From this result, we
p
have concluded that the oxidation of nitrite is diffusionꢀcontrolled process at RGO/[Cu(salꢀ
3
8
ala)(phen)]. Moreover, the electrocatalytic oxidation of nitrite at RGO/[Cu(salꢀala)(phen)]
modified electrode is an irreversible process and the anodic peak shifted to positive potential
when increasing the scan rate. The electron transfer coꢀefficient and the number of electrons
transfer involved in the rateꢀdetermining step can be estimated by the following equations
E = [2.303 RT/2 (1ꢀα ) n F ] log ν + K
(a)
(b)
pa
a
5
1/2
1/2 1/2
I = (2.99 × 10 ) [(1ꢀα)n ] AC × D
0
ν
p
n
a
0
9

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Where K is a constant, n is 1 and substituting the slope of E versus log ν plot, in the equation
a
pa
(a) the value of α was obtained as 0.52 for the nitrite oxidation. Furthermore, the number of
electrons involved in the oxidation of nitrite can be estimated by the equation (b) for totally
irreversible process controlled by diffusion. For equation (b), the D is the diffusion coefficient
0
of nitrite, A is the electrode area and C is the concentration of nitrite. By substituting all the
0
values in the above equation (b), the value of n estimated to be 2. This result indicated that the
–
nitrite oxidation lead to NO as a final product and it is good accordance with the literature. The
3
overall nitrite oxidation reaction can expressed by the equation (c) and (d)
ꢂ
ꢂ
ꢀ
ꢀNO − e ꢀ→ ꢀNO ꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢃcꢄ
ꢁ
ꢁ
ꢂ
ꢂꢀꢀ
ꢆ
ꢀ
ꢀ2NO +ꢀH Oꢀ ↔ NO ꢀ+ NO + ꢀ2ꢀH ꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢃdꢄꢀꢀ
ꢁ
ꢀꢀ
ꢁꢀ
ꢅ
ꢁ
From these results, the electro catalytic oxidation of nitrite at the RGO/[Cu(salꢀala)(phen)] nano
3
9
composite modified electrode as a two electron transfer reaction.
4.3 Amperometric determination of nitrite
Fig 7A shows the amperometric response of RGO/[Cu(salꢀala)(phen)] nanocomposite modified
rotating disc electrode (RDE) recorded at the rotation speed of 2000 rpm. The nitrite was
successively added in PBS with 50 s interval for every additions under the convection mode with
the applied working potential (E ) of +0.85 V. Herein, a sharp amperometric signal was
app
appeared for the determination of nitrite for the addition of 0.05 µM nitrite. Followed by that
addition, the concentration of nitrite was increased to 1000 µM. The resultant oxidation peak
current was linearly increased with increasing the concentration of nitrite (0.05 to 1000 µM).
From this result, we have observed two linear concentration ranges such as 0.05 – 4 µM (low)
and 6–1000 µM (high) for the determination of nitrite and the corresponding calibration plot was
shown in Fig 7B and C, respectively. The sensitivity of the fabricated sensor was calculated to be
–
2
3
.86 µA/µM cm and the lowest detection limit (LOD) calculated about 19 nM. The sensitivity
of the proposed sensor was compared with some other graphene based nitrite sensor. Notably,
the RGOꢀMWCNTꢀPt/Mb fabricated electrode was used for nitrite sensor with the sensitivity of
ꢀ2
0
.1651µA/µM/cm and GNPs/MWCNT modified electrode exhibit the sensitivity of about 2.58
ꢀ
2 40,41
µA/µM cm .
By the way, many of nitrite sensor electrodes were fabricated on the graphene
However,the RGO/[Cu(salꢀala)(phen)] nanocomposite modified electrode have
4
2ꢀ44
derivatives.
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ꢀ2
exhibited the high sensitivity (3.86 µA/µM /cm ) than that of the previously reported nitrite
sensor electrodes. Hence, it is evident that the Schiff base copper (II) complexes could be an
effective electrocatalyst for the oxidation of nitrite due to the higher electron transfer rate and
well redox properties. In addition, the high surface area of RGO provides strong interaction
between RGO and [Cu(salꢀala)(phen)]. The RGO/[Cu(salꢀala)(phen)] nanocomposite exhibited
the acceptable selectivity, wide linear range and low detection limit of nitrite. These observed
analytical results are compared with several others nitrite sensor electrode tabulated as shown in
Table 1.
4.4 Interference and real sample studies
We have analyzed the selectivity of RGO/[Cu(salꢀala)(phen)] nanocomposite modified electrode
towards the detection of nitrite in the presence of common interference ions such as anions,
cations and biological sample. The experimental condition was similar to in section 4.3 and the
corresponding amperogram shown in Fig 8A. The RGO/[Cu(salꢀala)(phen)] modified electrode
exhibited the well sharp amperometric response for the each 5 µM additions of nitrite (a).
Whereas, there is no response appeared for the 500 µM additions of other interfering ions, such
ꢀ
ꢀ
ꢀ
ꢀ
,
ꢀ
2+
2+
2+
2+
+
us (b) Br , (c) I , (d) Cl , (e) NO (f) F , (g) Cu , (h) Zn , (i) Cr , (j) Sr , (k) K , (l) dopamine,
3
(m) glucose, (n) fructose and (o) ascorbic acid. This study revealed that the RGO/[Cu(salꢀ
ala)(phen)] fabricated electrode selectively detect the nitrite even in the presence of excess
interfering compounds. Therefore, the RGO/[Cu(salꢀala)(phen)] nanocomposite applied to the
detection of nitrite in real time monitoring application. The practical feasibility of the
RGO/[Cu(salꢀala)(phen)] modified sensor has been demonstrated by amperometric method in
various sources of water samples and sausage meat sample. The water sample were collected
from the river and lake, the sausage sample was collected from local market in Taiwan. Before
performing the real sample analysis the collected water sample was subjected to the filtration and
remove the solid suspension. The standard addition method was used for the real sample analysis
and the obtained recoveries are 93.5 %, 94.3 % and 94.2 % is shown in Table 2. This result
endorsed that the developed nitrite sensor electrode has an acceptable recoveries in real sample
analysis. Thus, the modified electrode can be used for the determination of nitrite detection in
water and food samples.
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4.5 Repeatability, reproducibility and stability studies of RGO/[Cu(salꢀala)(phen)]
nanocomposite modified electrode
The repeatability and reproducibility of the proposed sensor was carried out by the CV studies in
the presence of 200 µM nitrite in PBS (pH 5). The modified electrode was exhibited an
acceptable repeatability with the RSD of about 3.55 % for the 10 repetitive measurements
examined by the single modified electrode. Moreover, the fabricated sensor electrode shows the
appreciable reproducibility of 3.45 % for the 10 measurements carried out by 10 different
modified electrodes. The obtained results showed that the RGO/[Cu(salꢀala)(phen)]
nanocomposite modified electrode have good repeatability and reproducibility. The storage
stability of the fabricated electrode was investigated periodically in PBS containing 200 µM
nitrite in an inert atmosphere. Fascinatingly, RGO/[Cu(salꢀala)(phen)] exhibited only 3.6 % of
peak current changes from its initial peak current response after the 30 days storage. The
operational stability of the RGO/[Cu(salꢀala)(phen)] nanocomposite modified electrode was
carried out by the amperometric technique in the presence of 50 µM nitrite for 3000 s (Fig 8B).
The experimental condition was similar as we discussed in the section 4.3 and manifest the
oxidized peak current loss only 2.2 % from the initial current. The obtained results reveals the
excellent operational stability and antifouling properties of the modified electrode.
5. Conclusions
In summary, we have successfully prepared a novel and stable RGO/[Cu(salꢀala)(phen)]
nanocomposite via nonꢀcovalent interaction and the prepared nanocomposite was characterized
by different microscopy and spectroscopic techniques. The RGO/[Cu(salꢀala)(phen)]
nanocomposite modified electrode showed an excellent electrocatalytic activity towards
detection of nitrite with wide linear concentration range (0.05ꢀ1000 µM), low detection limit (19
ꢀ2
nM) and high sensitivity (3.86 µA/µM cm ). The modified electrode offered an appreciable
repeatability and reproducibility. Moreover, the fabricated electrode was selectively determined
the nitrite even in the presence of interfering ions and biological molecules. Moreover, the
developed sensor electrode showed the good recoveries in real sample analysis from sausage
meat and water samples. Therefore, we concluded that the RGO/[Cu(salꢀala)(phen)]
nanocomposite modified electrode can be used as an excellent electrode material for the
electrochemical detection of nitrite. This proposed sensor electrode can be used in future for the
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accurate detection of nitrite in food and industrial samples. The reported Schiff base complex
have an advantages in terms of easy way to prepare, stable complex formation and higher
electrochemical activity. Thus, the Schiff base complex based nanomaterials have been extended
to develop a new electrochemical sensor.
Acknowledgement
The authors extend their appreciation to the International Scientific Partnership Program ISPP at
King Saud University for funding this research work through ISPP#6. Furthermore, this project
was supported by the National science council & Ministry of science and technology of Taiwan
(ROC).
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Figure captions,
Table 1 Comparison on the analytical performance of RGO/[Cu(salꢀala)(phen)] nanocomposite
modified electrode with other reported sensor.
Table 2 Determination of nitrite in real samples using RGO/ [Cu(salꢀala) (phen)] nanocomposite
modified electrode.
Scheme 1 Synthesis of [Cu(salꢀala)(phen)] inorganic complex.
Scheme 2 Schematic representation for the preparation of RGO/[Cu(salꢀala)(phen)]
nanocomposite for nitrite sensor.
Figure 1 SEM images of (A) GO, (B) RGO, (C) GO/[Cu(salꢀala)(phen)], (D) RGO/[Cu(salꢀ
ala)(phen)], (Inset C’) [Cu(salꢀala)(phen)], (E & F) EDX spectrum of RGO/[Cu(salꢀala)(phen)]
nanocomposite.
Figure 2 (A) Electrochemical impedance spectrum of (a) Bare GCE (b) GCE/RGO, (c) [Cu(salꢀ
ala)(phen)], (d) GO/[Cu(salꢀala)(phen)] and (e) RGO/[Cu(salꢀala)(phen)] modified electrode in
3
ꢀ/4ꢀ
0
.1 M KCl containing 5 mM [Fe (CN) 6] . (B) UVꢀVis spectroscopy analysis of (a) RGO, (b)
[
Cu(salꢀala)(phen)] and (c) RGO/[Cu(salꢀala)(phen)].
Figure 3 (A) Xꢀray diffraction studies of (a) RGO, (b) [Cu(salꢀala)(phen)], (c) RGO/[Cu(salꢀ
ala)(phen)]. (B) FTꢀIR spectra of (a) RGO, (b) [Cu(salꢀala)(phen)], (c) RGO/[Cu(salꢀala)(phen)].
Figure 4 (A) Cyclic voltammetry of (a) RGO/[Cu(salꢀala)(phen)] nanocomposite modified
electrode in absence of nitrite, (b) bare GCE, (c) GCE/GO, (d) GCE/[Cu(salꢀala)(phen)], (e)
GCE/RGO, (f) GO/[Cu(salꢀala)(phen)] and (g) RGO/[Cu(salꢀala)(phen)] in 0.05 M PBS (pH 5)
1
containing 200 µM nitrite at scan rate 50 mV s . (B) Cyclic voltammetry response of
RGO/[Cu(salꢀala)(phen)] nanocomposite at different concentration of nitrite in 0.05 M PBS (pH
ꢀ1
5
) at scan rate 50 mV s .
Figure 5 (A) Cyclic voltammetry response of RGO/[Cu(salꢀala)(phen)] nanocomposite modified
GCE in 200 µM nitrite containing different pH solution ( 2, 3, 5, 7, 9 & 11) at the scan rate of 50
ꢀ1
mV s , (5B) calibration plot for pH vs I .
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Figure 6 (A) Cyclic voltammetry response of the RGO/[Cu(salꢀala)(phen)] nanocomposite
modified GCE in PBS containing 200 µM of nitrite at different scan rate. (6B) calibration plot of
square root of scan rate Vs. peak current.
Figure 7 (A) Amperometric response for the oxidation of nitrite with different concentration in
PBS (pH 5), Eapp = 0.75 V. The calibration plot of peak current vs. nitrite concentration (low (B)
&
high (C))
Figure 8 (A) Amperometric response of hydrazine at RGO/[Cu(salꢀala)(phen)] in the presence
ꢀ
ꢀ
ꢀ
ꢀ
,
of 50 µM nitrite and 500 µM addition of different interferences (b) Br , (c) I , (d) Cl , (e) NO
3
ꢀ
2+
2+
2+
2+
+
(
f) F , (g) Cu , (h) Zn , (i) Cr , (j) Sr , (k) K , (l) dopamine, (m) glucose, (n) fructose and (o)
ascorbic acid. (8B) Stability studies of the RGO/[Cu(salꢀala)(phen)] nanocomposite modified
electrode.
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Table 1
a
Electrodes
Linear range (µM)
LOD (µM) References
b
c
d
RGO /[Cu(Salꢀala) (phen) ]
0.05ꢀ4, 6ꢀ1000
2.0ꢀ5000
0.019
2
This work
45
e
poly(methylene blue)/GCE
graphite/β –cyclodextrin
0.7ꢀ2150
3ꢀ5000
10ꢀ8000
3.6 ꢀ3.09
1ꢀ5000
5ꢀ180
0.26
1.12
33
46
47
48
49
50
51
52
53
54
55
f
Thionine modified aligned /CNT
g
RGOꢀZnO
h
i
j
Hb / PCAuPs /MWCNT
0. 96
2.4
k
AuNPS /GCE
l
CuO /GCE
1.6
m
PtNPs
10ꢀ1000
1ꢀ127
5
n
HAC
70
o
PANI conducting polymer
AuNP/Graphene/Chitosan
5ꢀ1400
1ꢀ380
0. 24
0.25
Abbreviation
a
b
c
d
e
Limit of detection, Reduced graphene oxide, salicylalanine, phenanthroline, Glassy carbon
f
g
h
I
electrode, carbon nanotubes, Zinc oxide, Hemoglobin, Positively charged gold nanoparticle,
j
k
l
m
Multiwalled carbon nanotubes,
Gold nanoparticles,
Copper oxide,
Platinum nano
n
o
particles, Heteroatom enriched porous carbon, Polyaniline
Table 2
a
Samples
Tap water
River water
Sausage 1
Sausage 2
Added (µM)
Found (µM)
9.56
Recovery (%)
RSD (%)
10
10
10
10
95.6
102
106
103
3.2
3.5
3.1
3.2
10.2
10.6
10.3
a
Relative standard deviation of 3 individual measurements
.
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Scheme 1
Scheme 2
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Figure 1
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Figure 2
Figure 3
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Figure 4
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3

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Figure 5
Figure 6
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Figure 7
Figure 8
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Graphical abstract,