Development of a biomimetic chitosan film-coated gold electrode for determination of dopamine in the presence of ascorbic acid and uric acid

Article abstract of DOI:10.1016/j.electacta.2010.06.062

A gold electrode surface was modified using a dinuclear copper complex [Cu^II₂ (Ldtb)(μ-OCH₃)](BPh₄) and then coated with a chitosan film. This biomimetic polymer film-coated electrode was employed to eliminate the interference from ascorbic acid and uric acid in the sensitive and selective determination of dopamine. The optimized conditions obtained for the biomimetic electrode were 0.1 M phosphate buffer solution (pH 8.0), complex concentration of 2.0 × 10^-4 M, 0.1% of chitosan and 0.25% of glyoxal. Under the optimum conditions, the calibration curve was linear in the concentration range of 4.99 × 10^-7 to 1.92 × 10^-5 M, and detection and quantification limits were 3.57 × 10^-7 M and 1.07 × 10^-6 M, respectively. The recovery study gave values of 95.2-102.6%. The lifetime of this biomimetic sensor showed apparent loss of activity after 70 determinations. The results obtained with the modified electrode for dopamine quantification in the injection solution matrix were in good agreement with those of the pharmacopoeia method.

Full text of DOI:10.1016/j.electacta.2010.06.062

Electrochimica Acta 55 (2010) 7152–7157
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Electrochimica Acta
journal homepage: www.elsevier.com/locate/electacta
Development of a biomimetic chitosan film-coated gold electrode for
determination of dopamine in the presence of ascorbic acid and uric acid
Suellen Cadorin Fernandesa,∗, Iolanda Cruz Vieiraa, Rosely A. Peraltab, Ademir Nevesb
a Departamento de Química, LaBios - Laboratório de Biossensores, Universidade Federal de Santa Catarina, CEP 88040-900, Florianópolis, SC, Brazil
b Departamento de Química, LABINC - Laboratório de Bioinorgânica e Cristalografia, Universidade Federal de Santa Catarina, CEP 88040-900, Florianópolis, SC, Brazil
a r t i c l e i n f o
a b s t r a c t
A gold electrode surface was modified using a dinuclear copper complex [CuII (Ldtb)(␮-OCH3)](BPh4)
Article history:
2
Received 31 March 2010
Received in revised form 21 June 2010
Accepted 21 June 2010
and then coated with a chitosan film. This biomimetic polymer film-coated electrode was employed to
eliminate the interference from ascorbic acid and uric acid in the sensitive and selective determination of
dopamine. The optimized conditions obtained for the biomimetic electrode were 0.1 M phosphate buffer
solution (pH 8.0), complex concentration of 2.0 × 10−4 M, 0.1% of chitosan and 0.25% of glyoxal. Under
the optimum conditions, the calibration curve was linear in the concentration range of 4.99 × 10−7 to
1.92 × 10−5 M, anddetection andquantificationlimits were3.57 × 10−7 Mand1.07 × 10−6 M, respectively.
The recovery study gave values of 95.2–102.6%. The lifetime of this biomimetic sensor showed apparent
loss of activity after 70 determinations. The results obtained with the modified electrode for dopamine
quantification in the injection solution matrix were in good agreement with those of the pharmacopoeia
method.
Available online 30 June 2010
Keywords:
Biomimetic complex
Gold electrode
Dopamine
Ascorbic acid
Uric acid
© 2010 Elsevier Ltd. All rights reserved.
1. Introduction
potentials, and thus different strategies have been used to mod-
ify the electrode surface, resulting in not only good sensitivity but
Dopamine (DA) is one of the most important natural cate-
cholamine neurotransmitters, playing an important role in the
functioning of the central nervous, renal and hormonal systems
[1]. Dysregulation of dopaminergic neurotransmission is associated
with attention deficit hyperactivity disorder, mood disorders and
schizophrenia [2].
Determination of DA is a subject of great importance and find-
ing selective methods for its quantification in the presence of high
levels of ascorbic acid (AA) and uric acid (UA) in body fluids is
relatively complicated [3]. Common instrumental techniques like
electrophoresis [4], chromatography [5], high performance liquid
chromatography [6–8] and spectrophotometry [9–11] have been
widely used for the determination of DA. However, such methods
of detection are often sophisticated and very expensive [12].
As DA shows very strong electrochemical activity, electrochem-
ical techniques appear to be strong candidates for its detection,
offering advantages such as simplicity, fast response and ease of
application. Most solid electrodes are associated with an over-
lapped signal, since the interferents may have close oxidation
also a high degree of selectivity [13–16]. One such strategy is to
employ a polymer-modified electrode that minimizes the access of
interferents through size exclusion, ion exchange and hydrophobic
interactions [17].
Chitosan is considered to be a promising material for modi-
fication of the electrode surface due to its attractive properties,
e.g., excellent film-forming ability, good stability, high permeability
toward water, strong adherence to the electrode surface, biocom-
patibility, no toxicity, high mechanical strength and susceptibility
to chemical modifications due to the presence of reactive amino
and hydroxyl functional groups [13,18].
Mimetic complexes have been used as simple models of natural
biochemical catalysts, mimicking their active site and their func-
tion as synthetic analogs, facilitating the electron transfer [19,20].
Additionally, they have the advantage of being more stable than
the natural enzyme under conditions of changes in temperature,
pH and solution composition, which often denature or inactivate
the natural biocatalysts [21].
Several groups have synthesized and characterized complexes,
which mimic the active site of enzymes [22–24], but very few use
them as biomimetic sensors for the oxidation of phenol substrates
[19–21,25–31]. Our group has studied a diversity of biomimetic
complexes developed by Neves and co-workers and which have
been successfully used to construct sensors [19,21,28–31]. These
include heterodinuclear FeIIIZnII [28] and FeIIIFeII complexes
[29], which mimic the active site of purple acid phosphatase, a
∗
Corresponding author at: Departamento de Química, LaBios - Laboratório de
Biossensores, Universidade Federal de Santa Catarina, Campus Universitário Reitor
João David Ferreira Lima, Bairro Trindade, CEP 88040-970, Florianópolis, Santa Cata-
rina, Brazil. Tel.: +55 48 3721 6844; fax: +55 48 3721 6850.
E-mail address: sucadorin@yahoo.com.br (S.C. Fernandes).
0013-4686/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.electacta.2010.06.062

S.C. Fernandes et al. / Electrochimica Acta 55 (2010) 7152–7157
7153
[N,N-Bis(2-pyridylmethyl)
butylbenzyl-2-hydroxy)(2-pyridylmethyl)]
aminomethyl]-6-[Nꢀ,Nꢀ-(3,5-di-tert-
homodinuclear MnIIIMnII complex of manganese peroxidase [19]
and tetranuclear [21] and dinuclear [30,31] copperII complexes,
that mimic the enzyme catechol oxidase. The dicopper complex
is one of a group of complexes with type 3 copper centers where
the metal ions are responsible for the catalysis of the oxidation of
o-diphenols to their corresponding o-quinones [32,33]. Dopamine,
which is a catechol-like phenolic compound, can be detected using
a catecholase-based system [34].
aminomethyl}-4-
methylphenol) was prepared by mixing 2-[bis-(2-pyridylmethyl)
aminomethyl]-6-(2-pyridylmethyl)aminomethyl-4-methylphenol
(3.4 g, 8 mmol) with triethylamine (1.21 g, 12 mmol) and 2-
chloromethyl-4,6-di-tert-butylphenol (3.02 g, 12 mmol) with
dichloromethane. The 1H NMR (CDCl3; ı ppm) data obtained for
this product were: 8.54, 7.61, 7.53, 7.15, 6.97, 6.84 (16H, aromatic
H), 3.92, 3.85, 3.84, 3.80 (s, 12H, CH2), 2.23 (s, CH3), 1.42 and 1.25
(s, t-butyl).
The aim of this study is to report an electroanalytical
methodology that combines a dinuclear complex [CuII2(Ldtb)(␮-
OCH3)](BPh4), which mimics the active site of catechol oxidase,
The complex [CuII2(Ldtb)(␮-OCH3)](BPh4) was obtained by
adding 1.0 mmol of Cu(OAc)2·H2O and 1.0 mmol of NaBPh4 and
Et3N, to a methanolic solution of 0.5 mmol of the ligand H2Ldtb,
under magnetic stirring, at 40 ◦C for 20 min. The crystal structures
were isolated for X-ray analysis, giving a product yield of 36%. Anal.
Calc. for Cu2C67H72BN5O3, M.W. = 1133.19 g mol−1: C, 71.0; H, 6.4;
N, 6.2. Found: C, 70.4; H, 6.0; N, 6.5%.
with
a chitosan film coating for the fabrication of a novel
biomimetic sensor. This sensor was constructed, optimized and
applied for determination of DA in pharmaceutical samples in the
presence of AA and UA. The results obtained with the proposed
biomimetic sensor compared favorably with those obtained using
the official method.
2. Experimental
2.4. Pretreatment of the gold electrode
2.1. Reagents
In the firstinstance, apretreatment procedure was applied to the
gold electrode surface (≥99.99% Au, 2 mm diameter, mounted in a
Teflon rod). Initially, the surface was polished mechanically with
alumina powder slurry on a polishing cloth for 2 min and rinsed
with deionized water. To remove the residual alumina particles, the
electrode was then ultrasonically cleaned in water and then ethanol
for 2 min in each case. The electrode was further treated by soaking
in a “piranha” solution (H2O2:H2SO4 = 1:3, v/v) for 10 min in order
to remove organic contaminants and subsequently washed with
deionized water. Finally, cyclic voltammograms were obtained in
All reagents were of analytical grade and all solutions were
prepared with a Millipore (Bedford, MA, USA) Milli-Q system
(model UV Plus Ultra-Low Organic Water). The gold electrode was
cleaned with alumina powder (0.3–0.05 ␮m) obtained from Aratec.
Ascorbic acid, uric acid, dopamine, sulfuric acid, hydrochloric
acid, ethyl alcohol, sodium acetate, sodium phosphate diba-
sic, sodium phosphate monobasic, acetic acid, glyoxal, chitosan
and tris(hydroxymethyl)aminomethane (tris) were obtained from
Sigma–Aldrich. Hydrogen peroxide, triethylamine (Et3N), sodium
tetraphenylboride (NaBPh4) and dichloromethane were pur-
chased from Merck. Inotropisa, labeled as containing 5.0 mg mL−1
dopamine, was purchased from a local pharmacy.
0.5 M sulfuric acid solution at between 0.0 and +1.7 V at 100 mV s−1
until reproducible voltammograms were obtained.
,
2.5. Preparation of the working electrode
2.2. Apparatus
The biomimetic sensor was prepared for film coating by depo-
sition of 5 ␮L of the [CuII2(Ldtb)(␮-OCH3)](BPh4) complex solution
(2.0 × 10−4 M) in dichloromethane and then left for solvent evapo-
ration at room temperature. Later, a chitosan film was obtained by
mixing 10 ␮L of 0.1% (m/m) chitosan solution with 3% acetic acid
solution together with another 10 ␮L of 0.25% (v/v) glyoxal solution.
With a micropipette, 10 ␮L of this mixture was placed directly onto
the gold electrode surface containing the complex and left to dry
overnight. The complex electrode was stored at room temperature
when not in use.
An ultrasonic bath (Unique, 1400A) with distilled water was
used to clean the electrode surface. The pH measurements were
taken with a Micronal B475 pH-meter using a combined glass elec-
trode.
Elemental analysis was performed with a Carlo Erba E1110 ana-
lyzer. IR spectra were obtained in the range of 4000–400 cm−1
with KBr pellets, using a Perkin-Elmer 781 spectrometer. 1H NMR
analysis of the ligand was carried out with a Bruker 200 MHz
spectrometer in CDCl3 chloroform, at 25 ◦C. Chemical shifts were
referenced to tetramethylsilane.
The cyclic voltammetry behavior of the complex was inves-
tigated in dichloromethane solution with a Princeton Applied
Research (PARC) model 273 potentiostat/galvanostat. The elec-
trochemical cell employed was of a standard three-electrode
configuration: platinum working electrode, platinum wire counter
electrode and Ag/AgCl reference electrode.
Voltammetric measurements for the dopamine determina-
tion were performed with an Autolab PGSTAT12 potentio-
stat/galvanostat (Eco Chemie, Utrecht, The Netherlands) driven
by data processing software (GPES, software version 4.9.006, Eco
Chemie). A conventional three-electrode cell was used, comprising
Ag/AgCl (3.0 M KCl) as the reference electrode, a platinum wire as
the counter electrode and a biomimetic modified gold sensor as the
working electrode.
2.6. Electrochemical measurements
Volumes of standard or sample solutions were added by
micropipette to the voltammetric cell containing 5.0 mL of the sup-
porting electrolyte (0.1 M phosphate buffer, pH 8.0) together with
ascorbic and uric acids when necessary. Square wave voltamme-
try (SWV) was performed in the range of +0.4 to −0.1 V vs Ag/AgCl
(3.0 M KCl) after 60 s of homogenization by stirring, applying a fre-
quency of 60.0 Hz, pulse amplitude of 100.0 mV and a step potential
of 7.0 mV. Samples were analyzed by the standard addition method
using the biomimetic gold electrode.
3. Results and discussion
3.1. Characterization of the dinuclear copper complex
2.3. Synthesis of the ligand (H2Ldtb) and the complex
[CuII2(Ldtb)(ꢀ-OCH3)](BPh4)
The ligand {2-[N,N-Bis(2-pyridylmethyl)aminomethyl]-6-
[Nꢀ,Nꢀ-(3,5-di-tert-butylbenzyl-2-hydroxy)(2-pyridylmethyl)]
aminomethyl}-4-methylphenol (H2Ldtb) was prepared in good
yields following the synthetic procedures described in Section
Peralta and co-workers have reported the synthesis of
[CuII2(Ldtb)(␮-OCH3)](BPh4) [33]. Firstly, the ligand H2Ldtb ({2-

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S.C. Fernandes et al. / Electrochimica Acta 55 (2010) 7152–7157
Fig. 1. Molecular structure of the complex cation [CuII2(Ldtb)(␮-OCH3)]+.
2. The complex [CuII2(Ldtb)(␮-OCH3)](BPh4) was prepared in
methanolic solution by adding Cu(OAc)2·H2O with NaBPh4 and
Et3N to the ligand H2Ldtb, yielding a dark red crystal. A schematic
representation of the dinuclear copper complex is shown in Fig. 1,
showing a pentacoordinated environment for both copper centers,
but with a significant distortion in the coordination environment
around Cu1, leading to an intermediate geometry between square
pyramidal and trigonal bipyramidal for Cu1, while Cu2 maintains
a square pyramidal geometry.
Fig. 3. Comparative cyclic voltammograms using (a) bare gold electrode, (b) chi-
tosan film-coated electrode, and (c) biomimetic chitosan film-coated electrode, in a
9.0 × 10−4 M dopamine solution in phosphate buffer (0.1 M; pH 8.0).
(0.1 M; pH 8.0): (a) with the bare gold electrode; (b) chitosan film
coated onto the gold surface and (c) the proposed biomimetic chi-
tosan film-coated electrode, in the potential range of +0.4 to −0.1 V
vs Ag/AgCl with a scan rate of 100 mV s−1
.
The cyclic voltammogram of [CuII2(Ldtb)(␮-OCH3)]+ in
dichloromethane (Fig. 2) solution shows only one irreversible
wave at −1.07 V vs Ag/AgCl as a consequence of the electronic
density of the deprotonated phenolate group. In addition to the
metal centered process, a quasi-reversible wave is apparent at
E1/2 = +0.27 V vs Ag/AgCl, attributed to a phenoxyl radical of the
ligand.
Peralta et al. [33] have reported other methods for the char-
acterization of this complex by UV–vis spectroscopy, magnetic
susceptibility, electron paramagnetic resonance and potentiomet-
ric titration.
Compared to the bare gold electrode, the current peak decreased
after coating the chitosan layer on the electrode surface, since it
acts as a mass transfer blocking layer, inhibiting the diffusion of
the dopamine towards the electrode surface. Despite this signifi-
cant decreased in the current response, the chitosan layer seems
to be important to eliminate the response of the concomitants
for dopamine determination. When the electrode surface is pre-
viously prepared with the biomimetic complex, a greater response
to the dopamine current was obtained. This indicates that the pres-
ence of the complex substantially improved the electron transfer
on the electrode surface. Catechol oxidases are responsible for the
oxidation of a variety of phenolic compounds to their correspond-
ing o-quinones with the simultaneous reduction of the oxygen to
water molecules. The [CuII2(Ldtb)(␮-OCH3)](BPh4) complex mim-
ics the action of catechol oxidase enzymes, oxidizing the dopamine
molecule to its corresponding o-quinone, which is electrochemi-
cally reduced at the electrode surface at a potential of +0.12 V, as
shown in Fig. 4.
3.2. Behavior of the modified electrode
To evaluate the contribution of the biomimetic complex and the
effect of the chitosan film on the electrode surface, different sen-
sors were constructed and compared using the cyclic voltammetry
technique. Fig. 3 shows their electrochemical performance in the
analysis of a 9.0 × 10−4 M dopamine solution in phosphate buffer
3.3. Optimization conditions of the biomimetic sensor
For dopamine determination using the biomimetic chitosan
film-coated electrode, several experimental conditions were eval-
uated: complex concentration, proportions of chitosan and glyoxal,
and pH, along with the frequency, pulse amplitude and scan incre-
ment used in the SWV. The response of the biomimetic sensor was
based on the resultant peak current for a 5.36 × 10−4 M dopamine
solution in 0.1 M phosphate buffer solution at pH 8.0 using SWV.
Initially, the effect of varying the copper complex concentration
from 2.0 × 10−3 to 2.0 × 10−5 M was investigated. The analytical
response of the modified sensor in 2.0 × 10−4 M CuIICuII complex
showed the highest resultant current. This complex concentration
was then used for further development of the biomimetic sensor.
Chitosan crosslinked with glyoxal was used to fix the complex
on the gold electrode surface and also to act as a selective poly-
mer modifier. Firstly, the glyoxal concentration was maintained
constant at 0.25% (v/v) and the chitosan concentration was var-
ied between 0.1, 0.5 and 1.0% (w/w) in 3.0% acetic acid solution.
The electrode containing 0.1% (w/w) of chitosan gave the high-
Fig. 2. Cyclic voltammogram for 1.0 × 10−3 M of the dinuclear copper complex in
dichloromethane and 0.1 M tetrabutylammonium hexafluorophosphate at a scan
rate of 100 mV s−1
.

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7155
Table 2
Result for recovery of dopamine standard solution from pharmaceutical injection
formulation using the proposed biomimetic chitosan film-coated electrode.
Dopamine (mg mL−1
Sample
)
Added
Founda
Recovery (%)
0.77
1.53
2.30
0.79 0.07
1.50 0.07
2.22 0.09
102.6
98.0
96.5
A
B
C
0.77
1.53
2.30
0.77 0.08
1.52 0.06
2.30 0.08
100.0
99.4
100.0
0.77
1.53
2.30
0.74 0.1
1.47 0.08
2.19 0.1
96.1
96.1
95.2
A–C, dopamine injection.
a
Standard deviation for three replicates.
(DA) are very close to each other and thus it is difficult to sep-
arate these compounds due to their overlapping signals [35,36].
This problem can be eliminated by electrostatic attraction, since a
chitosan film (pKa = 6.5), which is in its anionic form at the work-
ing electrode surface at pH 8.0, could attract DA molecules, which
are mainly in their cationic form with a pKa of 8.9. Also, AA and
UA undergo an electrostatic repulsion by the film since their pKa
values are 4.2 and 5.4, respectively, being in anionic form, as is
the chitosan. Table 1 shows a comparison between the potential
and resultant peak currents for a 1.99 × 10−6 M dopamine solu-
tion alone and in the presence of different concentrations of AA
and UA. On analyzing these values it can be concluded that the
presence of AA and UA did not interfere in the dopamine determi-
nation.
Also, different standard dopamine concentrations (0.77, 1.53
and 2.30 mg mL−1) were added to the samples followed by the cal-
culation of the percentage recovery to investigate the influence of
AA and UA when 1.99 × 10−4 M of these substances was added to
injection samples. The recoveries obtained for these samples were
95.2–102.6%, as shown in Table 2. It can be clearly observed from
the recovery results that AA and UA did not present interference in
the determination of DA.
Fig. 4. Schematic representation of a reaction involving dopamine on the surface of
the biomimetic sensor.
est response and was subsequently used in the construction of the
electrodes.
The percentage of chitosan was then maintained constant at
0.1% (w/w) and the glyoxal percentage was varied from 0.025 to
2.5% (v/v). The electrode containing 0.25% (v/v) offered a higher
response and this concentration was thus used to construct further
electrodes.
The effect of the different supporting electrolytes (acetate, phos-
phate and tris buffer solutions) and pH (5.0–9.0) on the sensor
response was investigated. The best voltammetric response was
obtained in phosphate buffer solution at pH 8.0. Therefore, these
conditions were used in further experiments.
The SWV parameters were optimized using the best response
of the biomimetic chitosan film-coated sensor to 5.36 × 10−4 M
dopamine solutions. Values of frequency in the range of 10–100 Hz,
pulse amplitude between 10 and 100 mV and scan increment from
1.0 to 12.0 mV were evaluated. The best resultant peak currents
were obtained using values for frequency, pulse amplitude and scan
increment of 60.0 Hz, 100.0 mV and 7.0 mV, respectively.
3.5. Calibration curve obtained in the electrochemical analysis of
dopamine
Under the optimum conditions, the SWV currents were
proportional to the dopamine concentrations over the range
of 4.99 × 10−7 to 1.92 × 10−5 M, giving a regression equation
of −ꢁi = 0.024( 0.006) + 6.032( 0.071) × 104 [Dopamine]; where
3.4. Selectivity and recovery study
It is well known that, using a bare gold electrode, the oxidation
peak potentials for ascorbic acid (AA), uric acid (UA) and dopamine
Table 1
Comparison of potential and resultant peak currents for 1.99 × 10−6 M dopamine in the presence of different concentrations of AA and UA.
E (V)
−ꢁi (␮A)
1.99 × 10−6
1.99 × 10−6
1.99 × 10−6
1.99 × 10−6
M
M
M
M
–
–
+0.12
+0.12
+0.12
+0.12
1.570 × 10−7
1.568 × 10−7
1.568 × 10−7
1.569 × 10−7
1.99 × 10−6
1.99 × 10−5
1.99 × 10−4
M
M
M
1.99 × 10−6
1.99 × 10−5
1.99 × 10−4
M
M
M

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S.C. Fernandes et al. / Electrochimica Acta 55 (2010) 7152–7157
Table 3
Analytical response using modified gold electrodes in dopamine determination.
Modification on gold electrode
Linearity (M)
Detection limit (M)
Reference
Corrole and self-assembled monolayers
Dopamine polymer film
Multi-walled carbon nanotubes
Cysteamine self-assembled monolayers
Self-assembled gold nanoparticle films
Fullerene-C60
Penicillamine self-assembled monolayer
3-Amino-5-mercapto-1,2,4-triazole
self-assembled monolayers
N-acetylcysteine self-assembled
monolayer.
dl-Homocysteine self-assembled
monolayers
Thiolactic acid self-assembled monolayer
l-Cysteine self-assembled monolayer with
peroxidase from beans sprouts
Biomimetic chitosan film-coated electrode
3.20 × 10−12–3.20 × 10−10
1.00 × 10−6–6.00 × 10−4
1.50 × 10−12
2.00 × 10−7
2.00 × 10−7
2.31 × 10−6
9.00 × 10−5
2.60 × 10−10
4.56 × 10−7
5.00 × 10−7
[12]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
5.00 × 10−7–4.00 × 10−4
6.00 × 10−6–3.84 × 10−4 and 3.36 × 10−4–9.50 × 10−3
2.00 × 10−4–1.20 × 10−3
1.00 × 10−6–5.00 × 10−3
8.06 × 10−6–1.06 × 10−3
1.50 × 10−6–1.00 × 10−4
1.00 × 10−6–2.00 × 10−4
5.00 × 10−6–5.00 × 10−4
8.00 × 10−7
5.00 × 10−7
[43]
[44]
4.00 × 10−5–8.00 × 10−4
2.37 × 10−6–3.76 × 10−5
3.00 × 10−6
2.95 × 10−6
[45]
[46]
4.99 × 10−7–1.92 × 10−5
3.57 × 10−7
This study
Table 4
Determination of dopamine (mg mL−1) in pharmaceutical formulation using the chitosan film-coated electrode.
Samples
Label value
Official methoda
Proposed electrodea
Er1 (%)
Er1 (%)
A
B
C
5.00
5.00
5.00
5.00 0.1
4.60 0.2
4.79 0.07
4.95 0.1
4.87 0.08
4.92 0.1
−1.0
−2.7
−1.6
−1.0
+5.5
+2.6
A–C, dopamine injection; Er1, proposed electrode vs label value; Er2, proposed electrode vs official method.
a
Standard deviation for three replicates.
−ꢁi is the resultant peak current in ␮A and [Dopamine] is the
dopamine concentration in M with a correlation coefficient of
0.9997. Fig. 5 shows these voltammograms and the analytical curve
can be seen in the inset. The detection limit (three times the
blank signal/slope) and quantification limit (10 times the blank
signal/slope) were found to be 3.57 × 10−7 M and 1.07 × 10−6 M,
respectively. We can find a number of modified gold electrodes for
dopamine detection in the literature. Table 3 compares the per-
formance of the sensor proposed in this study with that of other
electrodes, verifying that the biomimetic chitosan film-coated elec-
trode exhibits a linear range and limit of detection comparable
to those described in the literature, with the exception of two
cases.
3.6. Determination of dopamine using the proposed sensor
In order to evaluate the applicability of the proposed biomimetic
chitosan film-coated sensor, dopamine concentrations of pharma-
ceutical samples were determined. Prior to the measurements, the
dopamine injection samples were diluted in a 1:10 ratio with phos-
phate buffer solution (pH 8.0) and then used without previous
treatment. The results of the analysis were compared with those
obtained with the official USP method [47] as shown in Table 4, and
showed agreement at the 95% confidence level, within an accept-
able range of error. It can thus be concluded that this sensor is
suitable for application in the determination of dopamine with the
concomitant interferents.
3.7. Reproducibility, repeatability and stability of the proposed
sensor
The reproducibility for five biomimetic chitosan film-coated
electrodes was around 5.3% using a dopamine concentration of
1.99 × 10−6 M. The biomimetic sensor described herein offers a
highly reliable current signal for the repeatability of 8 measure-
ments for the same electrode with a relative standard deviation
(R.S.D.) of 3.7%. The long-term stability was investigated daily with
1.99 × 10−6 M dopamine solutions where the sensor was reused
for approximately 70 determinations showing 80% of activity loss
after one week. The stability of the proposed sensor can be ascribed
to the excellent immobilization of the complex in the chitosan
covalently crosslinked with glyoxal, which provides a microenvi-
ronment favorable for the entrapment of the biomimetic complex.
After 70 determinations the sensor response decreases probably
due to leaching of the complex as the sensor surface loses its chi-
tosan film.
Fig. 5. Square wave voltammograms obtained using the biomimetic sensor for (a)
blank in phosphate buffer solution (pH 8.0), and dopamine solutions at the fol-
lowing concentrations: (b) 4.99 × 10−7 M; (c) 9.98 × 10−7 M; (d) 1.99 × 10−6 M; (e)
4.95 × 10−6 M; (f) 9.80 × 10−6 M and (g) 1.92 × 10−5 M in 0.1 M phosphate buffer
solution (pH 8.0).
4. Conclusions
In this paper we report a simple, sensitive and selective electro-
analytical method using a biomimetic sensor based on a dinuclear

S.C. Fernandes et al. / Electrochimica Acta 55 (2010) 7152–7157
7157
CuIICuII complex coated with chitosan film for the electrochemical
determination of dopamine. This complex improved the oxidation
catalysis of dopamine enhancing the sensitivity of the biomimetic
sensor significantly. Also, the chitosan polymer layer was suit-
able for the immobilization of this complex and was successfully
employed as a good modifier for the selective determination of DA.
Hence, at a working pH of 8.0, the chitosan attracts DA (which
has a positive charge) and repels AA and UA (which have nega-
tive charges). The biomimetic chitosan film-coated electrode offers
an excellent linear calibration range, low detection limit, good
reproducibility and repeatability and facility of construction of
the sensor. The results demonstrated that dopamine was effec-
tively determined in the selected pharmaceutical samples using
this biomimetic sensor, when compared with the official method.
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