Colloids and Surfaces B: Biointerfaces 81 (2010) 110114

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Colloids and Surfaces B: Biointerfaces

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

Electrochemical behavior and analytical application of ciprooxacin using

a multi-walled nanotube composite lm-glassy carbon electrode
Lida Fotouhi , Mahnaz Alahyari
Department of Chemistry, School of Science, Alzahra University, P.O. Box 1993891176, Tehran, Iran

a r t i c l e

i n f o

Article history:
Received 1 May 2010
Received in revised form 24 June 2010
Accepted 29 June 2010
Available online 6 July 2010
Keywords:
Ciprooxacin
Multi-walled carbon nanotube
Cyclic voltammetry
Determination
Oxidation

a b s t r a c t
A simple, rapid and applicable electrochemical method was developed for the determination of
ciprooxacin (Cf) based on a multi-wall carbon nanotubes lm-modied glassy carbon electrode
(MWCNT/GCE). The constructed electrode (MWCNT/GCE) exhibited excellent electrocatalytic behavior
in the oxidation of Cf as evidenced by the enhancement of the oxidation peak current and the shift in the
oxidation potential to lower values (by 130 mV) in comparison with the bare GCE. A detailed analysis of
cyclic voltammograms and chronoamperograms gave fundamental electrochemical parameters including the electroactive surface coverage ( ), the transfer coefcient (), the standard rate constant (ks ) and
diffusion coefcient (D). Under optimized conditions in voltammetric method, the dynamic linear calibration curve for Cf was obtained in the concentration range of 401000 mol/L with the detection limit
of 6 mol/L. The analytical performance of this sensor has been evaluated for detection of the analyte in
urine and serum samples.
2010 Elsevier B.V. All rights reserved.

1. Introduction
Quinolones are a group of compounds widely applied in veterinary and human medicine. Ciprooxacin (Cf) [1-cyclopropyl-6uoro-1,4-dihydro-4-oxo-7-(piperazinyl) quinolone-3-carboxylic
acid) belongs to the second generation of quinolones with a uorine atom at the 6-position (uoroquinolones) and has a broad
spectrum of activity [1] together with an activity against several
intracellular bacteria [2].
These drugs form a group of antimicrobial agents used in the
treatment of urinary and respiratory tract infections with good
localized action on infected sites, gastrointestinal diseases as well
as in patients with intra-abdominal infections in combination with
antianaerobic agents [35]. These quinolones are effective against
gram-positive and gram-negative bacteria through interference
with enzyme DNA gyrase, which is needed for the synthesis of
bacterial DNA [6,7].
There is much interested in determining uoroquinolones for
the purpose of clinical and pharmaceutical quality control. Over
years, various techniques including high performance liquid chromatography (HPLC) [8,9], spectrophotometry [10,11], uorimetry
[12,13] have been proposed for the determination of Cf in pharmaceutical preparations or biological samples. Very few studies
involving electrochemical determination of Cf have been reported
[14,15].

Corresponding author. Tel.: +98 21 88044040; fax: +98 21 88035187.

E-mail addresses: lfotouhi@alzahra.ac.ir, lida fotouhi@yahoo.com (L. Fotouhi).
0927-7765/$ see front matter 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.colsurfb.2010.06.030

Carbon nanotubes are interesting class of nonmaterial offering

high electrical conductivity, high surface area, signicant mechanical strength and good chemical stability. They have been known to
promote electron transfer reactions when used as electrode modifying material. Utilization of these properties has led to application
of carbon nanotubes as scanning probes [16], electron eld emission sources [17], actuators [18], nanoelectronic devices [19] and
the electrocatalytic behavior toward many biomolecules [2025]
and drugs [26,27].
To our knowledge, electrochemical behavior and voltammetric
determination of Cf using MWCNT/modied glassy carbon electrode has not been reported yet. Following our recent studies
[2830] we herein report the voltammetric oxidation of Cf on multiwalled carbon nanotubes modied GCE. The effects of various
parameters on the electroactivity of MWCNT are also investigated.
Like electrochemical biosensors, this electrode exhibits high sensitivity, rapid response, good reproducibility, long-term stability and
freedom of other potentially interfering species. Finally this modied electrode is applied for the analysis of Cf in urine and serum
samples.
2. Experimental
2.1. Apparatus
Electrochemical measurements were carried out with Metrohm
model 746VA trace analyzer connected to a 747 VA stand. The
working electrode was a glassy carbon electrode (2 mm diameter).
Before use, the working electrode was sequentially polished with

L. Fotouhi, M. Alahyari / Colloids and Surfaces B: Biointerfaces 81 (2010) 110114

111

graded 10 M alumina powder, and rinsed with doubly distilled

water. A platinum wire and a commercial Ag/AgCl saturated KCl
from Metrohm were used as an auxiliary and reference electrodes,
respectively. The scan rate in cyclic voltammetry was 100 mV/s,
with exception of the experiments in which the inuence of this
variable was studied.
Chronoamperometric measurements were conducted using different concentration of Cf in solution at MWCNT-lm-modied
GCE. The initial potential (Ei ), where no electrolysis occurs and the
step potential (Es ) where complete electrolysis occurs, were 0.70
and 0.98 V, respectively (interval time,  = 50 s).
2.2. Reagents
Ciprooxacin was obtained from Sigma. Multi-walled carbon
nanotubes with purity 95% (1030 nm diameters and 5 m length)
were obtained from io-li-tec, Ionic Liquid Technologies. Solutions
were deaerated by bubbling high purity argon gas for 10 min
through them prior to the experiments. A stock BrittonRobinson
buffer solution 0.04 mol/L with respect to boric, orthophosphoric
and acetic acid was used in order to investigation of pH dependence of Cf. All other chemical reagents were of pro-analysis grade
from Merck and Fluka. All the solutions were prepared with doubly
distilled water.

Fig. 1. SEM image of the MWCNT/GCE.

peak potential at MWCNT/GCE was observed at less positive potential to be 0.97 V and the peak current increased compared with bare
GCE (Fig. 2, curve d). The increasing current as well as positive shift
of anodic peak demonstrate an efcient catalytic oxidation of Cf on
the MWCNT/GCE.

2.3. Preparation of the MWCNT-lm-modied GCE

3.2. Determination of electrochemical active surface area
The glassy carbon electrode was sanded in ultrane sand paper,
polished with 10 m alumina slurry in sequence and sonicated
successively in water and ethanol respectively for 10 min. 4 mg
MWCNT was added into 1 mL DMF. A homogeneous and stable
suspension of 4 mg/mL was achieved with the aid of ultrasonication agitation for about 20 min. The GCE was coated by casting 4 L
suspension of MWCNTDMF and dried in the air.

In order to obtain the electrochemical active surface areas

of both bare and modied electrode, the chronoamperogram
of 0.1 mmol/L potassium ferrocyanide as the redox probe was
recorded. In chronoamperometric studies, the current, i, for the
electrochemical reaction (at a mass-transfer-limited rate) of ferrocyanide which diffuses to an electrode surface is described by
Cottrell equation [32]:

2.4. Procedure for analysis of real samples

1 ml urine or serum sample was transferred to the electrochemical cell containing 9 ml of buffer solution, and the voltammograms
were recorded. The sample was spiked with certain amount of Cf
and the recovery of the spiked samples was determined.

i=

nFAD1/2 C
1/2 t 1/2

(1)

3.1. Electrochemical behavior of Cf at MWCNT/GCE

where A is the electrochemical active area, D is the diffusion coefcient, C* is the bulk concentration of ferrocyanide and the other
parameters have their typical meanings. Under diffusion control, a
plot of i vs. t1/2 will be linear and the value of A can be obtained
from the slope since the precise value of diffusion coefcient of ferrocyanide is well known (6.20 106 cm2 /s). The electrochemical
active area of MWCNT/GCE was 0.09 cm2 .

The distribution of MWCNT over the surface of modied glassy

carbon electrodes is shown by SEM (Fig. 1). As shown in Fig. 1,
porous MWCNT lm has large surface area. The SEM image also
reveals that the MWCNT are well distributed on the surface and
that most of the MWCNT are in the form of small bundles or single
tubes.
To evaluate the electrocatalytic behavior of MWCNT in Cf oxidation, the cyclic voltammograms of Cf were recorded at a bare
GCE and MWCNT/GCE in phosphate buffer (pH 7.0) (Fig. 2). In
the absence of Cf, no redox peaks was observed at both bare and
modied GCE electrodes during the cyclic voltammetry within the
potential window from 0.50 to 1.20 V (Fig. 2, curves a and b). Compared with the bare GCE (Fig. 2, curve a) a larger background
current was observed at the MWCNT-lm-modied GCE (Fig. 2,
curve b) during the cyclic voltammetric scan, which is probable
due to high double layer capacity [31]. Cyclic voltammogram of Cf
at bare GCE demonstrates a weak and broad irreversible oxidation
peak at 1.10 V (Fig. 2, curve c) due to slow electron transfer, while
the response was considerably improved at the MWCNT/GCE. The

Fig. 2. Cyclic voltammograms of (a) bare GCE in blank solution, (b) MWCNT/GCE in
blank solution, (c) bare GCE in the presence of 1 mmol/L Cf, (d) MWCNT/GCE in the
presence of 1 mmol/L Cf phosphate buffer (pH 7.0), scan rate: 100 mV/s.

3. Results and discussion

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L. Fotouhi, M. Alahyari / Colloids and Surfaces B: Biointerfaces 81 (2010) 110114

Scheme 1.

3.3. The effect of pH

equation [35] was used to estimate n and ks values:

The pH dependence of Cf was studied systematically in the pH

range from 2 to 10 in a BrittonRobinson buffer solution (Fig. 3).
As pH increased, Ep shifted toward less positive potentials due to
the hindrance of the oxidation at low concentrations of protons.
The formal E  is linear with respect to pH in the range of 27,
with a slope of 66 mV/pH (Fig. 3, inset). This value is close to the
theoretical value of 59 mV/pH [32] indicating the participation of
the same protons and electrons in the electrochemical process. The
electrochemical reaction process for Cf oxidation at MWCNT/GCE
can be summarized as shown in Scheme 1 [33].
The position of the break in the E  /pH plot implies that the
protonation site is associated with the electrode reaction, which
has an apparent pKa value of about 7.

Ep = E +

Useful information involving electrochemical mechanism can

usually be acquired from the relationship between peak current
and scan rate. Thus, the cyclic voltammograms of Cf at various
scan rates of 10, 20, 30, 40, 50, 60, 90, 100 and 150 mV/s were
recorded on the surface of the MWCNT/GCE (Fig. 4A). A linear
relationship (y = 19.857x + 320.39) with a correlation coefcient of
R = 0.9989 was observed between the peak current density and the
scan rate in 1090 mV/s, which revealed that the oxidation of Cf
was an adsorption-controlled process (Fig. 4B). Furthermore, from
the slope of the linear plot of current density vs.  the surface
concentration of electroactive species ( ) can be estimated about
5.3 108 mol/cm2 according to the following Eq. [34]:
n2 F 2 A
4RT

nF

ln

nF

ln 

(3)

where is the electron transfer coefcient, ks is the standard rate

constant of the surface reaction,  is the scan rate, n is the electron
transfer numbers and E is the formal potential. ks and n values
can be concluded from the intercept and slope of the linear plot of
Ep with respect to ln , if the E is known.
The E value at MWCNT-lm-modied GCE can be deduced from
the intercept of Ep vs.  plot on the ordinate by extrapolating the
line to  = 0 (Fig. 4C). By nding E , and graphical representations of
Ep vs. ln  for Cf in the presence of MWCNT (Fig. 4D), the n = 0.71
and ks = 177.9 s1 values were obtained from the slope and intercept, respectively.
3.5. Chronoamperometric studies

3.4. The effect of the scan rate

ip =

 RT    RTk 
s

Chronoamperometry can be used for determination of diffusion

coefcient, D, of Cf. We have obtained chronoamperograms at xed
potential of 0.98 V over 50 s in phosphate buffer (pH 7.0) containing
different concentrations of Cf (Fig. 5A).

(2)

As it is shown by increasing the scan rate, the peak potential is

shifted to more positive potential. Because of the irreversible electrode process of the oxidation reaction of Cf, the following Lavirons

Fig. 3. Cyclic voltammograms of 1.0 mmol/L Cf at MWCNT/GCE for pH values of

(right to left) (a) 2, (b) 3, (c) 4, (d) 5, (e) 6, (f) 7, (g) 8, (h) 9 and (i) 10 in
BrittonRobinson buffer solution, scan rate 100 mV/s. Inset, linear plot of E  vs.
pH at MWCNT/GCE.

Fig. 4. (A) Cyclic voltammetric responses of 1.0 mmol/L Cf at MWCNT/GCE phosphate buffer (pH 7.0) at scan rates, (inner to outer) 10, 20, 30, 40, 50, 60, 90, 100,
150 mV/s. (B) The plot of peak current density vs. scan rate. (C and D) The variation
of peak potential vs.  and ln , respectively.

L. Fotouhi, M. Alahyari / Colloids and Surfaces B: Biointerfaces 81 (2010) 110114

113

Fig. 6. The plot of current vs. Cf concentration.

Table 1
Results of analysis of real samples.

Fig. 5. (A) Chronoamperograms of Cf in phosphate buffer (pH 7.0) containing (a)

0.01, (b) 0.6, (c) 0.8, (d) 1.5 mmol/L Cf. (B) Plot of i vs. t1/2 for different concentrations.
(C) The plot of the slope of straight lines against the Cf concentration.

D values were estimated from the slope of a plot of i vs. t1/2

at different concentrations (Fig. 5B), according to Cotterll equation [32]. The slopes of the resulting straight lines were then
plotted vs. the Cf concentration (Fig. 5C). An average value of
D = 2.45 104 cm/s was obtained from this slope.
3.6. The effect of the amount and injected volume of MWCNT
composite
Optimization experiments were conducted to nd the optimum
conditions for MWCNT composite lm deposition with respect
to the voltammetric detection of Cf by varying the amount and
injected volume of MWCNT composite. The results showed that
peak current reached a maximum at 4 L of 4 mg of MWCNT composite and decreased beyond this concentration. At higher values
of MWCNT composite, a decrease in the signal was observed. This
decrease in the signal was probably due to a thicker MWCNT composite lm which is usually less stable accompanied with its lower
conductivity.
3.7. Analytical application, analytical gures of merit
Under optimum conditions, there was a linear relationship between peak current and Cf concentration in the range
401000 mol/L (Fig. 6). The limit of detection (LOD) was obtained
as YLOD = XB + 3SB , where YLOD is the signal for limit of detection,
and XB and SB are the mean and the standard deviation of the blank
signal, respectively [36]. Under optimum experimental conditions,
the LOD was obtained as 6.0 mol/L.
The reproducibility of the method was checked by successive determinations (n = 8) of Cf. The relative standard deviations
(R.S.D.) were lower than 1.0%. Also on using the MWCNT/GCE,
after leaving the electrode unused for a period of 2 weeks, the
peak potential for Cf oxidation remained constant and the cur-

Sample

Added (mg)

Found (mg)

Recovery (%)

R.S.D. (%) (n = 3)

Serum

1.00

0.009
0.928

91.97

1.19

Urine

1.70

0.11
1.67

92.36

1.62

rent signals showed only less than 5% decrease in the initial

response.
3.8. Interferences
The effects of some foreign species on the determination of
Cf were evaluated in detail. The effects of inorganic and organic
compounds commonly existed in pharmaceuticals and biological
samples on the determination of 0.4 mmol/L Cf were studied. The
tolerance limit was dened as the concentration ratio of additive/Cf
causing less than 5.0% relative error. 100-fold of Sr2+ , Cd2+ , Ba2+ ,
Cr2+ , Ni2+ , NH4 + , Ca2+ , Cu2+ , Mg2+ , Na+ , K+ , NO3 , SO4 2 , Cl , PO4 3 ,
acetate and 50-fold concentration of lysine, l-glutamic acid, l-serin,
l-histidine, urea, l-threonm and l-tyrosine, had almost no inuence on the current response of Cf (signal change below 5%). All
obtained results clearly represent the efciency of method for the
determination of Cf in the presence of the potentially interfering
species. The electrocatalytic effect of MWCNT in oxidation of Cf is
responsible for good selectivity observed on the proposed modied
electrode in the presence of the most important interferences. To
evaluate the applicability of the present method on real matrices,
the recovery of Cf was determined in serum and urine samples. The
results showed that satisfactory recovery for Cf could be obtained
(Table 1).
4. Conclusion
The present study indicates that the MWCNT/GCE exhibits good
electrocatalytic activity in Cf oxidation. The redox response of the
modied electrode is that anticipated for a surface-immobilized
redox couple. The electroactive surface coverage ( ), the transfer
coefcient (), standard rate constant (ks ) and diffusion coefcient
(D) were calculated from cyclic voltammetric and chronoamperometric responses. This modied electrode can be used for
voltammetric determination of Cf as low as 6.0 mmol/L with good
reproducibility and little fouling effects of Cf and its oxidation prod-

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L. Fotouhi, M. Alahyari / Colloids and Surfaces B: Biointerfaces 81 (2010) 110114

ucts. In addition, the results obtained in the analysis of Cf in urine

and serum samples demonstrated the applicability of the method
for real sample analysis.
Acknowledgment
The authors gratefully acknowledge partial nancial support
from the Research Council of Alzahra University.
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