Sensors and Actuators B 210 (2015) 483490

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Sensors and Actuators B: Chemical

journal homepage: www.elsevier.com/locate/snb

A sensitive electrochemical sensor with sulfonated graphene

sheets/oxygen-functionalized multi-walled carbon nanotubes
modied electrode for the detection of clenbuterol
Haiyun Zhai a, , Zhenping Liu a , Zuanguang Chen b, , Zhixian Liang a ,
Zihao Su a , Shumei Wang a
a
College of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, China
b
School of Pharmaceutical Science, Sun Yat-sen University, Guangzhou 510006, China

a r t i c l e i n f o a b s t r a c t

Article history: A novel electrochemical sensor was designed to determine clenbuterol by using a new hybrid nanoma-
Received 10 October 2014 terials of water-dispersible sulfonated graphene sheets (SGSs) and oxygen-functionalized multi-walled
Received in revised form carbon nanotubes (MWCNTs-COOH) modied glass carbon electrode (GCE). The stacking interac-
18 December 2014
tion between SGSs and MWCNTs-COOH was veried by UVvis spectroscopy, fourier transform-infrared
Accepted 30 December 2014
spectroscopy and scanning electron microscopy. The anodic peak current (Ipa) of clenbuterol at the
Available online 8 January 2015
GCE modied with SGSs/MWCNTs-COOH was much higher than those at the bare GCE, SGSs/GCE
and MWCNTs-COOH/GCE due to the synergistic effect of the modied nanomaterials. Differential
Keywords:
Sulfonated graphene sheets
pulse voltammetry disclosed a good linear relationship between Ipa and concentrations of clenbuterol
Oxygen-functionalized multi-walled (0.015.0 M), with the detection limit of 4.6 nM. Finally, the modied electrode was successfully used
carbon nanotubes to determine clenbuterol in liver samples.
Clenbuterol 2015 Elsevier B.V. All rights reserved.
Hybrid nanomaterials
Electrocatalyst

1. Introduction Therefore, it is necessary to put forward a sensitive method for the

Clenbuterol, a representative of synthesized 2-adrenergic ago-
nists, was originally developed for the treatment of pulmonary
diseases and asthma in humans and animals [1,2]. However, the
animals fed clenbuterol usually suffer from side effects such as
reduced fat levels and increased muscle protein anabolism. In addi-
tion, drug residue of clenbuterol may be prone to accumulation in
meat and body tissues for a long time, which causes acute poi-
soning after eating, giving rise to muscular pain, dizziness, cardiac
palpitation and vomiting [3]. Hence, 2-adrenergic agonists includ-
ing clenbuterol have been banned for animal production in most
countries including China to ensure safety [4]. The United Nations
Food and Agriculture Organization recommends a daily allowable
dose of 00.004 g kg1 for clenbuterol [5], and the European Union
has established maximum residue limits of 0.1 g kg1 in mus-
cles, 0.50 g kg1 in liver and kidneys, and 0.05 g kg1 in milk [6].
Corresponding author. Tel.: +86 020 39352129; fax: +86 20 3935 2129.
Corresponding author. Fax: +86 20 3994 3071.
E-mail addresses: zhaihaiyun@126.com (H. Zhai), chenzg@mail.sysu.edu.cn
(Z. Chen).
determination of clenbuterol.
Nowadays, different analytical methods, including liquid
chromatographymass spectrometry [7,8], gas chromatography
mass spectrometry [9,10], enzyme-linked immunoassay with poly-
clonal or monoclonal antibodies [11], capillary electrophoresis [12]
and electrochemical determination [13], have been developed to
detect clenbuterol in animal tissues, feedstuff and body liquids.
Although the methods are highly selective and sensitive for deter-
mining clenbuterol, the instruments per se as well as their running
and maintenance are often expensive. Moreover, the enzymes
which are intrinsically fragile always require relatively rigorous
temperature, pH, humidity and non-toxic chemicals [14,15]. Thus,
it is crucial to nd out inexpensive and facile methods for analyzing
clenbuterol.
Graphene, which comprises two-dimensional (2D) single or few
layers of sp2 -hybridized carbon atom sheets, has received huge
attention in electrochemical analysis since its discovery in 2004
because of unique nanostructure and extraordinary properties
[16]. Modied graphene oxide (GO) sheets such as sulfonated
graphene sheets (SGSs) and partially reduced GO sheets are supe-
rior to ordinary GO ones. Recently, graphene-based materials have
also been utilized as catalysts and biosensors. Graphene-based

http://dx.doi.org/10.1016/j.snb.2014.12.121
0925-4005/ 2015 Elsevier B.V. All rights reserved.
484 H. Zhai et al. / Sensors and Actuators B 210 (2015) 483490
nanocomposites such as polymer/graphene, metal oxides/
graphene, partly reduction graphene and SGSs have been suc-
cessfully designed and used due to the interaction between
graphene sheets [17,18].
Carbon nanotubes (CNTs), which were discovered by Iijima in
1991, have been highlighted as electrochemical sensors owing to
unique structures and excellent properties [19]. However, CNTs are
not easily soluble in a large number of solvents, thus signicantly
limiting their manipulations and applications, especially in the
fabrication of CNTs-modied electrodes. Hence, various func-
tionalized CNTs have been synthesized in order to facilitate the
suspension in aqueous solution. For example, Shi et al. investigated
the antioxidant behavior of hydroxylated multi-walled carbon
nanotubes (MWCNTs) in high density polyethylene [20]. Korani
and Salimi prepared an amine-derivative MWCNTs-modied elec-
trode based on the covalent attachment of glucose dehydrogenase
and safranin O [21].
In this study, we employed a stable aqueous dispersion of
hydrophilic SGSs/MWCNTs-COOH hybrid nanomaterials to prepare
a modied electrode for the rst time. The electrochemical behavior
of clenbuterol was evaluated using the electrode, and the catalytic
mechanisms of clenbuterol at the electrode were investigated. The
modied electrode provided a large electrochemically active sur-
face area for the catalysis of clenbuterol and effectively accelerated
the electron transfer between the electrode and solution, which
allowed a more rapid and sensitive current response. Besides, the
modied electrode exhibited better sensitivity and selectivity for
clenbuterol detection in liver samples with satisfactory recoveries.
2. Experiment
2.1. Reagents and apparatus
Hydrazine (98 wt% in water) and graphite powder (99%, 40 nm)
used for synthesizing GO and SGSs were obtained from Aladdin
Inc. (Shanghai, China). MWCNTs (95%, 1020 nm in diameter and
1030 m in length) used for synthesizing oxygen-functionalized
MWCNTs (MWCNTs-COOH) were purchased from DKnano Co., Ltd.
(Beijing, China). Clenbuterol was purchased from National Insti-
tutes for Food and Drug Control (Beijing, China). Phosphate buffer
solutions (PBS) with pH ranging from 5.0 to 10.0 were prepared by
mixing stock solutions of 0.2 M NaH2 PO4 and 0.2 M Na2 HPO4 , and
pH was adjusted with H3 PO4 (1.0 M) and NaOH (1.0 M) solutions.
Unless otherwise stated, all the other chemicals were of analyti-
cal grade and purchased from Guangzhou Chemical Reagent Co.,
Ltd. (Guangzhou, China). Deionized water was used to prepare the
aqueous solutions.
Cyclic voltammetry (CV) and differential pulse voltammetry
(DPV) were performed on a CHI 850 C electrochemical workstation
(CH Instrument, Shanghai, China). A conventional three-electrode
system was used for all electrochemical experiments. Bare or
modied glass carbon electrode (GCE, = 3 mm) acted as the
working electrode, and an Ag/AgCl-saturated KCl electrode and
a platinum electrode were used as the reference electrode and
the auxiliary electrode (Gaoss Union, Wuhan, China), respectively.
UVvis absorption spectroscopy was conducted with a Shimadzu
2550 probe spectrophotometer (Kyoto, Japan). Fourier-transform
infrared (FT-IR) spectrum was obtained on a Mattson Cygnas 100
FT-IR spectrometer (Shelton, USA). The surface of the modied elec-
trode was examined by a scanning electron microscope (SEM), and
images were obtained from a Quanta 400F thermal eld emission
environmental FEI-SEM (Philips, Amsterdam).
2.2. Preparation of MWCNTs-COOH and SGSs modied materials
MWCNTs-COOH were synthesized according to a previously
reported method [22]. In a typical experiment, 75.0 mL of H2 SO4
(98%) and 25.0 mL of HNO3 (65%) were mixed and added to 1.0 g of
MWCNTs in a round-bottomed ask and reuxed under constant
circumuence at 50 C for 8.0 h. After the experiment, MWCNTs-
COOH were obtained, and then washed to neutral by deionized
water. The residue was then ltered and vacuum-dried.
Water-dispersible SGSs were synthesized from graphite pow-
ders. Three steps were involved in this process. Initially, GO was
synthesized according to the modied Hummers method reported
by Kovtyukhova et al. [23]. The obtained GO was dispersed in deion-
ized water, yielding a dark brown suspension. Secondly, sodium
borohydride was used to reduce GO. Thereafter, as reported pre-
viously by Li et al. [15], a black suspension was prepared by 4 h of
reaction with aryl diazonium salt of sulfanilic acid in an ice bath
that kept the temperature below 5 C. Thirdly, 98 wt% hydrazine
was added into the dispersion. The mixture was kept at 100 C for
24 h under stirring to complete the reduction. The powders were
obtained by being centrifuged, washed with deionized water sev-
eral times and dried at 60 C for 24 h.
The hybrid nanomaterial (SGSs/MWCNTs-COOH) was prepared
according to a previous literature with slight modications [24].
Particularly, 5.0 mg SGSs were mixed with 5.0 mg formal MWCNTs-
COOH in 10 mL deionized water and sonicated for 2 h. The
resulting suspension was then centrifuged so that the as-prepared
SGSs/MWCNTs-COOH hybrid nanomaterial could be well dispersed
in water and stored as a 0.5 mg mL1 aqueous solution.
2.3. Preparation of the SGSs/MWCNTs-COOH modied electrode
Prior to modication, the prepared GCE ( = 3 mm) was
mechanically polished twice with 0.05 m -Al2 O3 to a mirror n-
ish. Thereafter, the polished electrode was rinsed with deionized
water, sonicated in HNO3 (1:1), dehydrated alcohol and deion-
ized water successively, and dried in air at room temperature.
Then 10 L of the hybrid SGSs/MWCNTs-COOH dispersion was
cast on the electrode surface with a micro-syringe and the sol-
vent was evaporated at room temperature in air to obtain the
SGSs/MWCNTs-COOH/GCE. Before each measurement, the modi-
ed electrode was rinsed with PBS (0.1 M) and then scanned from
0.2 V to 1.0 V, and the analyte adsorbed on the modied electrode
surface after measurement was readily removed by soaked in the
KOH (0.5 M) solution for half a minutes.
2.4. Preparation of samples
Pork liver samples were purchased from a local supermar-
ket, and were pretreated according to previous literatures [25,26].
Briey, 1.0 g smashed sample was homogenized using 2 mL of 0.1 M
HClO4 , ultrasonicated for 20 min, and then heated at 80 C for
30 min. After cooling and 15 min of centrifugation at 3000 rpm, the
clear liquid phase was collected. Then pH of the collected liquid
was adjusted to 910 by using 1.0 M Na2 CO3 , into which 1.6 g NaCl
was then added. Subsequently, clenbuterol was extracted twice
by using 20 mL of ethyl ether. Finally, clenbuterol was reversely
extracted by 2 mL of 0.1 M HCl solution. The reverse extraction was
repeated, and the sample solution was diluted to 10 mL by using
pH 7.0 PBS.
3. Results and discussion
3.1. Characteristics of SGSs/MWCNTs-COOH/GCE
The improvement in the physicochemical properties of the
SGSs/MWCNTs-COOH composite depends on the distribution of
SGSs in the MWCNTs-COOH bundles as well as the interfacial bond-
ing between the SGSs and MWCNTs-COOH bundles. The surface
 H. Zhai et al. / Sensors and Actuators B 210 (2015) 483490
Fig. 1. SEM images of SGSs (A), MWCNTs-COOH (B) and SGSs/MWCNTs-COOH hybrid nanomaterials (C).
morphology of SGSs/GCE, MWCNTs-COOH/GCE, SGSs/MWCNTs-
COOH/GCE was characterized using SEM, and the images were
shown in Fig. 1. SGSs (Fig. 1A) consisted of large sheets with slightly
scrolled edges, and the crumpled sheets formed a disordered solid
via close interactions. The folded regions of the sheet were approxi-
mately 5 nm thick in average. MWCNTs-COOH were entangled with
each other to give a network structure with the average diameter
of about 20 nm (Fig. 1B). Crumpled, rippled SGSs and long, tortu-
ous MWCNTs-COOH formed a homogeneous hybrid nanomaterial
(Fig. 1C), possibly through the stacking interaction between
the hydrophobic region of SGSs and the sidewalls of MWCNTs-
COOH [27,28].
Fig. 2A presents the UVvis spectra of GO (a), SGSs (b) and
SGSs/MWCNTs-COOH (c). The UVvis spectrum of GO dispersion
exhibits two characteristic absorption peaks. The strong band at
232 nm corresponds to the * transition of C C, and the shoulder
peak at 299 nm can be assigned to the n* transition of C O [29].
After reduction by the methods herein, the absorption peak red-
shifted to 261 nm and the shoulder peak disappeared, accompanied
by the color change from brown to dark (inset of Fig. 2A). Moreover,
the absorption peak of SGSs/MWCNTs-COOH bathochromically
shifted to 271 nm, indicting strong interaction between them.
The FT-IR spectrum of GO (Fig. 2B) displays well-dened charac-
teristic peaks at  (CO) = 1050 cm1 ,  (CC) = 1615 cm1 and 
(CO) = 1733 cm1 . After reduction, the  (CO) = 1733 cm1 band
disappeared. Moreover, there were peaks at  (SO) = 1119 cm1
and  (SO) = 1168 cm1 , demonstrating the successful reduction
of GO to SGSs nanomaterials [15,30].
CV is an effective and convenient technique for the determina-
tion of current response. Fig. 2C shows the cyclic voltammograms
485
of 5.0 mM potassium hexacyanoferrate (K3 Fe(CN)6 ) solution
containing 0.1 M potassium chloride (KCl) at different electrodes
(bare GCE, MWCNTs-COOH/GCE, SGSs/GCE and SGSs/MWCNTs-
COOH/GCE). The redox process of K3 Fe(CN)6 was reversible at the
bare GCE (curve a). When the electrode was modied with 10 L
(0.5 mg mL1 ) of MWCNTs-COOH or SGSs suspension, the redox
peak currents increased conspicuously (curves b and c), which
can primarily be attributed to the enlarged effective surface and
the augmented current signal. When the hybrid SGSs/MWCNTs-
COOH dispersion was immobilized on the GCE (curve d), the current
response of the system reached maximum. Probably, the electro-
chemical properties were related to the electrocatalytic activity of
SGSs/MWCNTs-COOH that exerted a synergistic effect on the con-
ductivity of the sensor. According to the RandlesSevcik Eq. (1)
[31]:
Ip = 2.69 105 n3/2 Aeff D1/2 v1/2 C (1)
where Ip is the peak current (A), n is the number of electrons
transferred, Aeff is the effective area (cm2 ), D is the diffusion
coefcient of 5.0 mM K3 Fe(CN)6 and 0.1 M KCl (cm2 s1 ), v is the
scan rate (V s1 ) and C corresponds to the bulk concentration
of the probe (mol cm3 ). Then the apparent electroactive sur-
face area of SGSs/MWCNTs-COOH/GCE is calculated as 0.087 cm2 ,
which exceeds those of MWCNTs-COOH/GCE (0.056 cm2 ) and
SGSs/GCE (0.066 cm2 ). As indicated by the increases of estimated
active surface area and background current, SGSs/MWCNTs-COOH
hybrid nanomaterials could remarkably improve the electrochemi-
cal properties of the modied electrode and accelerate the electron
transfer.
486 H. Zhai et al. / Sensors and Actuators B 210 (2015) 483490

Fig. 2. A. UVvis absorption spectra of GO (a), SGSs (b) and SGSs/MWCNTs-COOH hybrid nanomaterials (c). Inset: photograph of GO aqueous solution (right) and SGSs
dispersion (left). B. FT-IR spectra of GO (a), SGSs (b) and SGSs/MWCNTs-COOH (c). Inset: enlarged image of the FT-IR spectra plot for SGSs under the region of 10001300 cm1 .
C. Cyclic voltammograms of 5.0 mM K3 Fe(CN)6 and 0.1 M KCl at bare GCE (a), MWCNTs-COOH/GCE (b), SGSs/GCE (c) and SGSs/MWCNTs-COOH/GCE (d) at a scant rate of
0.05 V s1 .

3.2. Electrochemical behaviors of clenbuterol at different
electrodes
Fig. 3 exhibits the cyclic voltammograms of different electrodes
in the presence of 10.0 M clenbuterol. The anodic peak currents
(Ipa) and potentials (Epa) of clenbuterol at different modied elec-
trodes were recorded. The bare GCE (curve a) gave practically no
voltammetric response to clenbuterol, demonstrating that the elec-
trochemical behavior of clenbuterol remained stable in the studied
potential window. Since only one anodic peak corresponding to the
electrochemical oxidation of clenbuterol was obtained in potential
of 0.41.0 V at MWCNTs-COOH/GCE, SGSs/GCE and SGSs/MWCNTs-
COOH/GCE, the processes at them were irreversible. After SGSs or
MWCNTs-COOH suspension was cast on the surface of GCE, Ipa
rose apparently at SGSs/GCE (curve c) and MWCNTs-COOH/GCE
(curve b), which can mainly be ascribed to the carbon nanoma-
terials on the electrode surface. Moreover, the Ipa responses at
SGSs/GCE were much higher than those at MWCNTs-COOH/GCE,
suggesting that SGSs allowed better electrode transfer for clen-
buterol than MWCNTs-COOH did because of higher conductivity
and larger surface area. Besides, compared with the Ipa at bare
GCE, SGSs/GCE and MWCNTs-COOH/GCE, the response Ipa of clen-
buterol at SGSs/MWCNTs-COOH/GCE increased signicantly (curve
d) owing to the synergistic effect of MWCNTs-COOH and SGSs
incorporated in the lm. For one thing, SGSs gaps were linked with
the MWCNTs-COOH network. SGSs adhered to the MWCNTs-COOH
bundles, thus enlarging the surface area of the modied electrodes.
For another, SGSs/MWCNT-COOH hybrid nanomaterials consider-
ably adsorbed clenbuterol by hydrogen bonding, hydrophobic force
and electrostatic interaction due to the negative charge introduced
by sulfonic acid.

3.3. Optimization of analytical conditions for clenbuterol

3.3.1. Effect of buffer pH

Fig. 3. Cyclic voltammograms of 10.0 M clenbuterol at bare GCE (a), MWCNTs-
COOH/GCE (b), SGSs/GCE (c) and SGSs/MWCNTs-COOH/GCE (d) in 0.1 M PBS at a
scant rates of 0.05 V s1 .
The effect of pH of the supporting electrolyte on the clen-
buterol (10.0 M) electrooxidation at SGSs/MWCNTs-COOH/GCE
was investigated (Fig. 4A) by altering peak currents and peak
potentials. The Ipa of clenbuterol increased gradually with the
increasing pH from 5.0 to 7.0, and attained maximum value
at pH 7.0. Furthermore, the Epa of clenbuterol shifted to neg-
ative, being linearly related with the solution pH from 5.0 to
10.0. The relationship between Epa and pH was described by
Epa/V = 0.0532 pH + 1.1848 (R = 0.9937). According to the rela-
tionship: dEp/dpH = 2.303mRT/nF [32], where m and n refer to
the transferring number of proton and electron, respectively. The
number of m/n in this reaction was calculated to be 0.899, which
 H. Zhai et al. / Sensors and Actuators B 210 (2015) 483490
Fig. 4. A. Cyclic voltammograms of 10.0 M clenbuterol at SGSs/MWCNTs-COOH/GCE in 0.1 M PBS at different pH (from left to right): 10.0, 9.0, 8.0, 7.0, 6.0 and 5.0, respectively.
Insets: plot of Ipc versus pH and plot of Epa versus pH. B. Effect of accumulation time on Ipa of 10.0 M clenbuterol. C. Effect of concentration of SGSs/MWCNTs-COOH on Ipa
of 10.0 M clenbuterol.
indicated that an equal number of electrons and protons are
involved in the oxidation of clenbuterol [33], this result agreed with
the reported by Lin et al. [14].
3.3.2. Effect of accumulation time
SGSs/MWCNTs-COOH/GCE was investigated from 1 to 8 min to
determine the optimum accumulation time. Ipa response obviously
increased with increasing incubation time and reached a platform
at 6 min (Fig. 4B), presenting that the accumulation equilibrium
was obtained at the electrode interface. Therefore, considering
sensitivity and analysis, 6 min was chosen as the experimental
accumulation time.
3.3.3. Effect of concentration of SGSs/MWCNTs-COOH suspension
To optimize the electrochemical response of clenbuterol, differ-
ent concentrations of SGSs/MWCNT-COOH suspension were cast
onto the GCE surface (Fig. 4C). The current response of clen-
buterol was maximum with 0.5 mg mL1 SGSs/MWCNTs-COOH
suspension. The reason might be that SGSs/MWCNTs-COOH hybrid
nanomaterials raised the accumulation efciency and peak cur-
rent by evidently enlarging the active area of the GCE surface.
Subsequently, a small amount of SGSs/MWCNTs-COOH hybrid
nanomaterials will notably enhance the oxidation response of clen-
buterol [18,34]. Conversely, the peak current plummeted with
further increase amount of SGSs/MWCNTs-COOH suspension. Since
SGSs/MWCNTs-COOH were densely packed on the electrode sub-
strate, the effective surface decreased and thus the electrical
conductivity of the nanomaterials reduced. Therefore, 0.5 mg mL1
SGSs/MWCNTs-COOH suspension was chosen as the optimum con-
centration to modify the GCE surface.
3.3.4. Effect of scan rate
Fig. 5A shows the cyclic voltammograms of 10.0 M clen-
buterol at different scan rates of SGSs/MWCNTs-COOH/GCE. At low
487
potentials (00.4 V), a pair of asymmetrical redox peaks (Ia and Ic)
appeared with increasing scan rate, revealing an irreversible oxi-
dation process of clenbuterol. In addition, the peak currents were
proportional to the scan rates in the range of 0.010.4 V s1 (Fig. 5B),
with the calibration equations of Ipa (A) = 258.6v 3.687 (V s1 )
(R = 0.9968) and Ipc (A) = 359.0v 5.523 (V s1 ) (R = 0.9935).
Hence, the electrochemical process of clenbuterol at lower
potentials was surface-controlled process. However, at high poten-
tials (0.81.0 V, IIa), Ipa rose linearly with increasing square
root of scan rate (Fig. 5C), with the calibration equations of
Ipa (A) = 74.20v1/2 3.879 (V s1 ) (R = 0.9964). Accordingly, the
oxidation of clenbuterol at high potentials at SGSs/MWCNTs-
COOH/GCE was a diffusion-controlled process. Additionally, Epa
shifted to positive with increasing scan rate. Moreover, Epa was lin-
early related with the natural logarithm of scan rate (lg v) at high
potential as shown in Fig. 5D. According to the Lavirons theory
[35], for an irreversible anodic reaction, the relationship between
Epa and v can be described by Eq. (2):
RT RTk RT
Ep = E  + ln + ln v (2)
nF nF nF
where E is the formal potential (V), is the charge transfer coef-
cient, n is the number of transferred electrons, and k is the standard
heterogeneous electron transfer rate constant. R, T and F have their
conventional meanings. In this study, Epa increased linearly with
lg v ranging from 0.08 to 0.4 V s1 following Eq. (3):
Epa = 0.01033 lg v + 0.9403 (R = 0.9965) (3)
Combining Eq. (2) with Eq. (3), n was calculated as 0.49.
According to a previous study, it is usual that is assumed as 0.5 for
a totally irreversible electrode progress [2,36]. Hence, an electron
was involved in the oxidation of clenbuterol, being consistent with
the results previously reported [37]. As suggested by the relation-
ship between Epa and pH, identical numbers of proton and electron
488 H. Zhai et al. / Sensors and Actuators B 210 (2015) 483490
Fig. 5. A. Cyclic voltammograms of 10.0 M clenbuterol in 0.1 M PBS (pH 7.0) at SGSs/MWCNTs-COOH/GCE at various scan rates (from inner to outer: 0.010.40 V s1 ). B.
The relationship of the Ipa and Ipc on scan rates (v) at low potential (00.4 V). C. The relationship of the Ipa and the square root scan rates (v1/2 ) at high potential (0.81.0 V).
D. The relationship of the Epa and the logarithm of scan rates (lg v) at high potential (0.81.0 V).
were transferred. Therefore, the oxidation of clenbuterol involved
a proton and an electron [25] (Scheme 1).
Besides the amount of clenbuterol on the electrode surface ( )
can be evaluated form the relationship between currents and scan
rate (Iv) can be described by Eq. (4) [38]:
n2 F 2 vA
Ip =
4RT
where v is the scan rates (V s1 ) and A is the surface area (cm2 ), R,
T, F are formal or have their usual values. Assuming, the number of
electrodes transferred n as 1, then  of clenbuterol was calculated
to be 4.34 1010 mol cm2 .
3.4. Determination of clenbuterol by DPV
Under the optimized conditions mentioned above, DPV was
selected to sensitively determine clenbuterol at SGSs/MWCNTs-
COOH/GCE. Fig. 6A shows the differential pulse voltammograms of
0.015.0 M clenbuterol in 0.1 M phosphate buffer (pH 7.0) with
applied potentials of 0.40.9 V. Ipa was linearly proportional to
the concentration of clenbuterol, with the linear regression equa-
tion of Ipa (A) = 1.231C + 0.0563 (M, R = 0.9996) (Fig. 6B). The
detection limit of clenbuterol at the SGSs/MWCNTs-COOH modied
Scheme 1. Oxidation mechanism of clenbuterol at SGSs/MWCNTs-COOH/GCE.
electrode was 4.6 nM, which is superior to some of those the elec-
trodes reported previously (Table 1) [13,29,3942].
3.5. Repeatability, reproducibility, stability and interference
The repeatability, reproducibility and stability of the sen-
(4) sor were evaluated by CV under the optimum conditions. One
SGSs/MWCNTs-COOH/GCE was evaluated by determining 10.0 M
clenbuterol ve times. Given that the relative standard deviation
(RSD) was 2.7%, SGSs/MWCNTs-COOH/GCE was highly repeatable.
The stability of the modied electrode was evaluated using ampero-
metric technique for 60 min of continuous operation. Ipa of 10.0 M
clenbuterol at SGSs/MWCNTs-COOH/GCE was studied by keeping
Epa constant at 0.82 V, and the current response retains more than
99.3%. Moreover, ve modied electrodes prepared independently
managed to determine 10.0 M clenbuterol with RSD of 3.7%. After
the electrodes were kept at room temperature for half a month,
the Ipa of 10.0 M clenbuterol was still as high as 93.2%. Thus, the
modied electrode was also highly stable.
Under the optimized conditions, the changes of Ipa were mea-
sured in the presence of different concentrations of potential
interferences. Even 1000-fold K+ , Ca2+ , Cl , NO3 and SO4 2 as well
as 500-fold uric acid, glucose, terbutaline, and salbutamol failed to
affect the detection of 1.0 M clenbuterol. Therefore, this sensor
was highly selective for clenbuterol.
3.6. Analysis of matrix spiked samples
In order to evaluate the feasibility of the newly developed sensor
for the determination of clenbuterol in liver samples, the prepa-
ration of sample was described in Section 2.4. Each sample was
measured seven times. No clenbuterol was detected in these sam-
ples. And then three different concentrations of clenbuterol were
spiked in the liver samples by using the standard addition method.
The results of each sample and recovery of each spiked sample
 H. Zhai et al. / Sensors and Actuators B 210 (2015) 483490 489

Fig. 6. Differential pulse voltammograms of SGSs/MWCNTs-COOH/GCE in different concentrations of CLB in 0.1 M PBS (pH 7.0). Inset a: 0.01, 0.03, 0.05, 0.08, 0.1 and 0.2 M
CLB, Inset b: linear relationship of Ipa and concentrations.

Table 1
Comparison of the fabricated sensor with reported voltammetric methods for the detection of CLB.

Modied electrode Technique Detection limit (M) Linear range (M) Ref.

Naon-Au DPV 0.1 0.810 [13]

AuNPs/CPE DPSV 0.0092 0.01760.527 [29]
Gold ultra microelectrode CV 0.002 0.0010.1 [39]
Poly taurine/ZrO2/GCE LSV 0.02 5220 [40]
Hybrid carbon nanotubes/GCE DPV 0.05 0.133 [41]
Chitosan-MWCNTs/GCE DNPV 0.08 0.540 [42]
SGSs/MWCNTs-COOH/GCE DPV 0.0046 0.015.0 This work

Table 2 bare GCE, SGSs/GCE and MWCNTs-COOH/GCE, the SGSs/MWCNTs-

Determination of CLB in liver (n = 7).
COOH/GCE exhibited enhanced electrocatalytic activity towards
Technique Added (M) Found (M) Recovery (%) RSD (%) clenbuterol. The oxidation of clenbuterol involved an electron and
This work 0.1 0.096 96.0 3.6 a proton. Under the optimized experimental conditions, the linear
0.5 0.517 103.4 2.3 range was 0.015.0 M for clenbuterol, which could be applica-
1.0 0.983 98.3 3.3 ble for real samples determination. Thus, a sensitive, selective
HPLC 0.1 0.104 104.0 1.8 and reproducible and cost-effective electroanalytical method was
0.5 0.510 102.0 1.1
developed for the discrimination and measurement of clenbuterol.
1.0 0.994 99.4 2.1
Ergo, the new hybrid nanomaterials are potentially eligible for sen-
sors, nanoelectronics, and electrochemical applications.
were calculated, and the values were listed in Table 2. Also given
in this table were the results achieved by the HPLC method [43].
The HPLC conditions included separation on an Ecosil EPS-C18 col- Acknowledgements
umn (250 mm 4.6 mm, 5 m particle size), the mobile phase was
methanol0.2% aqueous formic acid (60:40, v/v, pH 4.0) with a The authors gratefully acknowledge nancial support from the
ow rate of 1.0 mL min1 , column thermostat was set at 25 C, National Natural Science Foundation of China (NSFC, Grant No.
and detecting wavelength was set at 214 nm. The analytical data 21005021 and No. 21375152) Guangdong Provincial Science and
for various samples given in Table 2 exhibited a high degree of Technology Project (No. 2011112 and No. 2010B0203106010).
correlation between the results of high performance liquid chro-
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Biographies
Haiyun Zhai received her Ph.D. in analytical chemistry from Sun Yat-sen University
of China in 2005. As an associate professor of the Department of Pharmaceutical
Analysis, College of Pharmacy, Guangdong Pharmaceutical University, China, her
current research interests are instruments of capillary electrophoresis, miniaturiza-
tion, electrochemical sensor, sample pre-treatment technology and pharmaceutical
analysis.
Zhenping Liu is presently pursuing his Master degree in pharmaceutical analysis
from Guangdong Pharmaceutical University of China. His current research focuses
on electrochemical sensor and its application in pharmaceutical analysis.
Zuanguang Chen received his Ph.D. and B.S. degrees in analytical chemistry from
Sun Yat-sen University of China in 2000 and 1982, respectively. As a professor and
director of the Institute of Analytical Instrument, School of Pharmaceutical Sciences,
Sun Yat-sen University, China, his current research interests include instruments of
capillary electrophoresis and microuidic system, and pharmaceutical analysis.
Zhixian Liang is presently pursuing his Master degree in pharmaceutical analysis
from Guangdong Pharmaceutical University of China. His current research focuses
on electrochemical sensor and its application in pharmaceutical analysis.
Zihao Su is also presently pursuing his Master degree in pharmaceutical analy-
sis from from Guangdong Pharmaceutical University of China. His current research
focuses on sample pre-treatment technology and its application in pharmaceutical
analysis.
Shumei Wang received her Ph.D. in the traditional Chinese of medicine from Beijing
University of Chinese Medicine in 2007. As a professor and vice-president of College
of traditional Chinese medicine, Guangdong Pharmaceutical University, China, her
current research interests are pharmaceutical analysis and basic materials research
of traditional Chinese medicine.