Electroanalytical properties of haemoglobin in silica-nanocomposite films electrogenerated on pyrolitic graphite electrode
Haemoglobin (Hb) modified electrochemical devices have been prepared by Hb encapsulation in silica sol-gel films (SiO2), which were generated by electro-assisted deposition onto pyrolitic graphite electrodes (PGEs). The stability and electrocatalytic activity of Hb entrapped into SiO2 network was substantially enhanced in the presence of cationic surfactant (CTAB) and Au nanoparticles (Au-NPs). The composition of sol-gel synthesis medium, i.e., molar ratio of silica precursor to water, contents of Hb, CTAB and Au-NPs, as well as the conditions of electrogeneration had a great influence on the electrocatalytic activity of Hb on PGE surface. The electrochemical response of the PGE modified with the composite SiO2-Hb-CTAB-Au-NPs film was found to vary linearly with the concentration of dissolved oxygen in solution and this was exploited to determine this analyte in the tap water with detection limit 0.12 mg L-1. The electrocatalytic current of dissolved oxygen was also found to decrease in the presence of the antivirus drug--amino derivative of adamantane (rimantadine)--which opens the way to the determination of this drug with detection limit 0.3 mg L-1 using PGE modified with SiO2-Hb-CTAB-Au-NPs nanocomposite film.
Electroanalytical properties of haemoglobin in silica-nanocomposite ﬁlms
electrogenerated on pyrolitic graphite electrode
, O. Tananaiko
, I. Mazurenko
, M. Etienne
, A. Walcarius
, V. Zaitsev
Department of Analytical Chemistry, National Taras Shevchenko University, Volodymyrska 64, Kyiv 01601, Ukraine
Laboratoire de Chimie Physique et Microbiologie pour l’Environnement, UMR 7564, CNRS-Nancy University, 405 rue de Vandoeuvre, 54600 Villers-les-Nancy, France
Received 21 May 2008
Received in revised form 4 September 2008
Accepted 6 October 2008
Available online 17 October 2008
Haemoglobin (Hb) modiﬁed electrochemical devices have been prepared by Hb encapsulation in silica
sol–gel ﬁlms (SiO
), which were generated by electro-assisted deposition onto pyrolitic graphite elec-
trodes (PGEs). The stability and electrocatalytic activity of Hb entrapped into SiO
network was substan-
tially enhanced in the presence of cationic surfactant (CTAB) and Au nanoparticles (Au-NPs). The
composition of sol–gel synthesis medium, i.e., molar ratio of silica precursor to water, contents of Hb,
CTAB and Au-NPs, as well as the conditions of electrogeneration had a great inﬂuence on the electrocat-
alytic activity of Hb on PGE surface. The electrochemical response of the PGE modiﬁed with the compos-
–Hb–CTAB–Au-NPs ﬁlm was found to vary linearly with the concentration of dissolved oxygen in
solution and this was exploited to determine this analyte in the tap water with detection limit
0.12 mg L
. The electrocatalytic current of dissolved oxygen was also found to decrease in the presence
of the antivirus drug––amino derivative of adamantane (rimantadine)––which opens the way to the
determination of this drug with detection limit 0.3 mg L
using PGE modiﬁed with SiO
Au-NPs nanocomposite ﬁlm.
Ó2008 Elsevier B.V. All rights reserved.
Amperometric biosensors based on enzymes entrapped in silica
matrices coated on solid electrode surfaces become increasingly
popular as this way of biomolecule immobilization imparts high
stability and good catalytic activity [1–3]. Sol–gel-derived silica-
based materials are indeed attractive hosts for enzymes as they
are chemically inert towards biomolecules and their three-dimen-
sional structure does not restrict conformation mobility of the lat-
est, so encapsulated biomolecules can retain their catalytic activity
[4–6]. On the other hand, it was shown earlier that heme proteins
such as haemoglobin (Hb) and myoglobin acquire peroxidase
activity while immobilised [7,8] and haemoglobin-based ampero-
metric biosensors were proposed for determination of various ana-
lytes, including O
[9–11]. The detection scheme
involved the catalytic reduction of these compounds on the Hb-
modiﬁed electrodes. Another promising application of Hb-modi-
ﬁed electrodes is the determination of some pharmaceuticals
(e.g., antivirus or anticancer drugs) that are likely to interact with
Hb. Examples are available for detection of Taxol and ribavirin
using Hb-modiﬁed electrodes on the basis of a decrease in the cat-
alytic current relative to O
reduction upon increasing the drug
Recently the method of electrodeposition was proposed to ob-
tain thin sol–gel coatings on solid electrode surfaces, which was
achieved via an electrochemical modulation of pH at the elec-
trode/solution interface to promote geliﬁcation of the sol in a con-
trolled way [14,15]. Electrodeposition method shows a few
advantages comparing to classic spin-coating and dip-coating
methods, particularly the ability to control the thickness of the
ﬁlms, greater porosity and simplicity of procedure . This was
notably applied to get organically-functionalized silica thin ﬁlms
on electrodes [16–18] or well-structured and oriented deposits
[19,20], which can be applied as sensitive nano-layers with good
analytical performance [21,22]. Very recently, we have demon-
strated that such an electro-assisted generation approach can be
extremely useful for encapsulation of biomolecules, i.e., Hb and
glucose oxidase, in silica thin ﬁlms deposited on glassy carbon
electrodes . The encapsulated enzymes retained their catalytic
activity without additional use of mediators. Still the problem of
the stability of immobilised proteins was not fully solved, giving
notably rise to signiﬁcant decrease in the analytical signal with
time, what we have tried to circumvent in the present study by
the use of surfactant and/or nanoparticles additives.
The addition of cationic surfactant (for example cetyltrimethyl-
ammonium bromide, CTAB) at CMC and higher level as template
agent into silica sol permits to obtain well-structured porous mate-
rials [24,25]. Surfactants indeed improve the properties of sol–gel
silica derived materials where encapsulated organic molecules are
0022-0728/$ - see front matter Ó2008 Elsevier B.V. All rights reserved.
*Corresponding author. Tel.: +38 0 44 239 34 44; fax: +38 0 44 239 33 45.
E-mail address: firstname.lastname@example.org (O. Tananaiko).
Journal of Electroanalytical Chemistry 625 (2009) 33–39
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dispersed more uniformly in the silica matrix due to their favour-
able interaction with the micelles of surfactant formed inside silica
cage . It is known that surfactants can form biomembrane-like
structures which are disposed to entrap protein molecules like Hb.
Such approach was used earlier to construct carbon electrode mod-
iﬁed by cationic surfactant and hemoprotein . CTAB prevents
leaching of the protein molecules from the silica network and does
not inﬂuence the catalytic activity of encapsulated enzymes [8,28].
Non-ionic surfactant Tween 20 was also proposed to promote
immobilisation of Hb onto the pyrolitic graphite electrode in the
form of self-assembled multilayer ﬁlm .
On the other hand, the addition of metal nanoparticles, i.e., gold,
into biocomposite materials can greatly enhance the electrochem-
ical properties of ﬁlm electrodes containing encapsulated enzymes.
Nanoparticles act somewhat as wires between the active centre of
the enzyme and the electrode surface, thus providing facile elec-
tron transport and effective transduction of the biochemical recog-
nition event [30,31]. This approach was successfully applied to the
development of mediator-free glucose biosensors based on glucose
oxidase absorbed on colloidal gold or encapsulated into a gold
nanoparticles–mesoporous silica composite [32,33]. The electrodes
modiﬁed with gold nanoparticles were also used for further mod-
iﬁcation with heme-proteins (Hb  and peroxidase ) and
successfully applied for development of biosensors. The fast aggre-
gation of gold nanoparticles in the silica sol can be achieved with
the help of various types of stabilizers. Biomolecules themselves
can be used as stabilizer of gold nanoparticles in solutions when
containing thiol groups in their structure [31,36].
In the present work we have thus examined various approaches
to improve the stability of Hb molecules encapsulated into sol–gel
silica ﬁlms which were electrogenerated onto the surface of pyro-
litic graphite electrode. The effect of the biocomposite composi-
tion, and especially the addition of surfactants molecules and/or
gold nanoparticles in the starting silica sol, was thoroughly studied
with respect to the electrocatalytic properties of encapsulated Hb.
Furthermore, the Hb-modiﬁed electrode was tested for the deter-
mination of dissolved oxygen and an antivirus drug based on the
amino derivative of adamantane (rimantadine).
2.1. Chemicals and solutions
Human haemoglobin (Hb, Mw 64,000) was purchased from Sig-
ma. Tetraethoxysilane (TEOS, 98%) was obtained from Fluka.
Cetyltrimethylammonium bromide (CTAB, Sigma) and polyoxyeth-
ylene derivative of sorbitan monolaurate polysorbate-20 or Tween
20 (Merck) were used as additives in the sol–gel synthesis proce-
dure as required. All solutions were made up with double-distilled
water. All other chemicals were of analytical grade and used with-
out further puriﬁcation. HCl (0.01 mol L
) was used for precursor
hydrolysis. The 0.07 mol L
phosphate buffer solutions were pre-
pared by mixing stock solutions of KH
justed at selected pH values using either HCl or NaOH.
The suspension of Au nanoparticles was prepared according to
the standard procedure . Brieﬂy: 1% HAuCl
was reduced by
1% aqueous solution of sodium citrate. The dimension of colloidal
particles in aqueous suspension and in the silica sol was controlled
spectrophotometrically at k= 510–520 nm [38,39]. Water or silica
sol was used as blank solution.
The mixture of Hb and Au was prepared by addition of Hb solu-
tion to Au nanoparticles suspension in various Au:Hb volume ra-
tios (Au:Hb = 4:1; 3:1; 2:1; 0.4:1). The pH of the mixture was
adjusted to 10 by NaOH. The solutions were stored at 4 °C when
not in use.
The concentration of dissolved oxygen in water samples was
determined using the standard Winkler titration method .
The solutions with different concentration of dissolved oxygen
were prepared by mixing the solution with known oxygen content
and oxygen-free solution at different volume ratio. The oxygen free
solution was obtained of purging nitrogen through the 15 mL of
water for 10 min. All solutions were tightly closed before and dur-
The solution of rimantadine was prepared by dissolving one pill
of Remavir (OlainFarm, Latvia) containing 50 mg of rimantadine
hydrochloride in 15 mL of ethanol. Then the solution was diluted
with phosphate buffer pH 6.0. The content of rimantadine in solu-
tions was controlled by LC–MS method using as a standard ethanol
solution of the substance of the known concentration similar to
[41,42] but without previous derivatization of the substance. Be-
fore voltammetric measurements the content of dissolved oxygen
in rimantadine solutions was measured using the standard Win-
kler titration method. All solutions were tightly closed during the
experiments to avoid contact with air.
2.2. Preparation of Hb-modiﬁed electrodes
Pyrolitic graphite electrode (PGE, basal plane, ‘‘Burevestnik”, St.
Petersburg, Russia) was modiﬁed with silica Hb-containing ﬁlms
by electrogeneration technique by adapting a procedure described
earlier for glassy carbon electrodes . PGE was ﬁrst polished
with the help of diamond paste, washed with ethanol and water,
and dried at 90 °C for 1 h. The thin sol–gel ﬁlms were electrogen-
erated on the clean PGE surface from an aqueous TEOS-based sol
solution to which the Hb solution was added. A typical silica sol
was prepared by dissolving 2.125 g TEOS, 2 mL of water and
2.5 mL of 0.01 mol L
aqueous HCl, which were mixed for 12 h
using a magnetic stirrer. Then 0.07 mol L
phosphate buffer solu-
tion (pH 6.0) was added to silica sol to increase pH and a necessary
volume of the protein solution (0.5 mmol L
) was added to the
hydrolyzed sol. The mixture was introduced into the electrochem-
ical cell where electro-assisted generation was performed at
1.2 V at room temperature for 7 s (optimized time). After the thin
Hb-containing silica ﬁlm was formed on the surface of PGE (PGE
–Hb), the electrode was rinsed with water, dried for 60 min
in air and stored at 4 °C if not in use. To optimize the ﬁlm compo-
sition, selected amounts of CTAB was added into the sol solution
just before electro-assisted generation and the so-called Hb–sil-
ica-surfactant composite ﬁlm electrodes were denoted PGE SiO
Hb–CTAB. Au nanoparticles-doped Hb-containing nanocomposite
ﬁlms were obtained by adding the mixture of Hb and Au solutions
(volume ratio 1:2) to the silica sol prior to applying the electrogen-
eration procedure, in the absence or in the presence of CTAB,
leading to the formation of PGE SiO
–Hb–Au and PGE SiO
Electrogeneration and voltammetry experiments have been car-
ried out using analytical voltammeter AVA-2 (‘‘Burevestnik”, St.
Petersburg, Russia). Measurements were performed at room tem-
perature in a three-electrode cell, including the modiﬁed PGE
working electrode, an Ag/AgCl reference and a Pt wire auxiliary
electrode. Most often, the electrolyte solutions were deoxygenated
by bubbling nitrogen for 10 min prior to the experiments. The
ionometer I-130 was used for pH monitoring. Spectrophotometer
SF-46 (Severodonetsk, Russia) with quarts cuvette l= 1 cm was
used for absorption spectroscopic measurements.
The composite electrodes were analyzed by atomic force
microscopy (AFM) using a commercial microscope (Thermomicro-
scope Explorer Ecu+, Veeco Instruments S.A.S.) to get information
34 T. Rozhanchuk et al. /Journal of Electroanalytical Chemistry 625 (2009) 33–39
of the ﬁlm morphology. Scanning electrochemical microscopy
(SECM) was further used to visualize the inﬂuence of conducting
nanoparticles in the thin layers on the interface reactivity. The set-
up has been built-up in our laboratory (LCPME, Nancy, France) on
the base of the apparatus commercialized by Sensolytics (Bochum,
Germany). The approach curve experiments have been performed
with a 25
m platinum disc electrode in a solution
containing 0.1 mol L
KCl and 1 mmol L
The sample position (z= 0) has been estimated by comparing the
experimental approach curve with the corresponding calculated
curve (Rg 5) . All approaches have been stopped automati-
cally at a constant distance from the surface by use of a shear-force
3. Results and discussion
3.1. Voltammetric characteristics of SiO
–Hb modiﬁed PGE
Fig. 1 shows cyclic voltammogram of PGE and PGE SiO
the absence or presence of oxygen, as recorded at 100 mV s
phosphate buffer solution (pH 6.0). In the absence of oxygen, the
response of PGE SiO
–Hb was characterized by two peaks, a well-
deﬁned cathodic signal located at 0.20 V (versus Ag/AgCl) (E
and its (less visible) anodic counterpart at 0.06 V (versus Ag/
), as illustrated by curve 2 in Fig. 1 (enlarged in the inset).
These peaks were ascribed to reduction and oxidation of iron in
porphyrinic complex of Hb molecules. The peak potentials of qua-
si-reversible Fe(III)/Fe(II) couple on the modiﬁed PGE were posi-
tively shifted comparing to those measured on GCE . In the
presence of oxygen the cathodic peak current dramatically in-
creased, with potential shifting to 0.3 V (versus Ag/AgCl), which
refers to catalytic reduction of O
, whereas the anodic part van-
ished (Fig. 1, curve 3). For the bare PGE no peaks were observed
in the mentioned potential range (Fig. 1, curve 1). The obtained
data demonstrates that Hb molecules in the SiO
–Hb ﬁlm on the
PGE surface were electroactive and possessed catalytic activity.
Electrogeneration parameters such as deposition time and ap-
plied potential inﬂuence the ﬁlm thickness, its permeability to
external reagents and its mechanical stability and thereby the vol-
tammetric response of the modiﬁed electrode [17,18]. The effect of
these two parameters was studied here for Hb-containing sol solu-
tions by varying the applied potential between 1.0 and 1.4 V
and deposition times ranging from 5 s to 60 s. The highest electro-
catalytic currents, I
, for oxygen response at PGE SiO
observed when applying a deposition potential of 1.2 V and
deposition time of 7 s. These optimum values are very close to
those reported for similar ﬁlms deposited on glassy carbon
=1.2 V and t
The amount of Hb in silica sol was found to inﬂuence the value
of the modiﬁed electrode. I
of PGE SiO
–Hb increased lin-
early with raising of Hb concentration in silica sol from 5
up to 40
and reached saturation at the Hb content of
But still the catalytic activity of Hb immobilized on PGE was not
stable as I
values decreased rapidly upon successive analyses and
disappeared after 18 potential sweeps.
3.2. Inﬂuence of surfactant addition in silica sol on the properties
of the Hb-modiﬁed PGE
Cationic (CTAB) and non-ionic (Tween 20) surfactants were
studied as additives likely to improve the performance of the Hb-
containing electrogenerated silica ﬁlms. In the absence of oxygen,
no detectable difference in cyclic voltammograms of PGE SiO
Hb–CTAB and PGE SiO
–Hb was observed, indicating that CTAB
did not affect the electrochemical response of encapsulated Hb.
In the presence of oxygen, however, the electrocatalytic signal
was found to increase a little bit and to shift in the positive direc-
tion (by ca. 50 mV) when using PGE SiO
–Hb–CTAB instead of PGE
–Hb (compare curves 1 and 2 in Fig. 2). This demonstrates eas-
ier reduction of O
at the electrode modiﬁed with biocomposite in
the presence of CTAB. On the other hand, this advantage belonging
to surfactant was not observed with Tween 20, which resulted in
signiﬁcant decrease in catalytic activity of encapsulated Hb possi-
bly due to fast aggregation of biomolecules in silica sol. It was in-
deed checked that silica sols containing Hb and Tween 20 were not
stable and precipitation of the protein was observed within 30 min
so that Tween 20 was not used in further investigation.
Addition of CTAB to sol–gel mixtures not only improved the
electrocatalytic response of the biocomposite ﬁlm electrode but
also increased it upon successive voltammetric measurements
(compare curves 1–3 in Fig. 3 and especially curves 1 and 2 in
Fig. 4). For example, the value of I
for PGE SiO
creased by 50% after 22 potential sweeps while for PGE SiO
completely disappeared after 18 sweeps (Fig. 4, curves 1 and 2).
The composition of the starting sol, notably the CTAB concentra-
tion and TEOS:H
O ratio, was found to affect signiﬁcantly the per-
Fig. 1. Cyclic voltammetric curves recorded in 0.01 mol L
phosphate buffer (pH
6.0) and 0.01 mol L
at PGE (1) and PGE SiO
–Hb (2–3) obtained in anaerobic
(2) and aerobic (1 and 3) conditions at a scan rate of 100 mV s
voltammetric curve 2.
Fig. 2. Voltammetric curves recorded under aerobic conditions at PGE SiO
–Hb–CTAB (2), PGE SiO
–Hb–Au (3) and PGE SiO
–Hb–CTAB–Au (4). Other
conditions as in Fig. 1.
T. Rozhanchuk et al. /Journal of Electroanalytical Chemistry 625 (2009) 33–39 35
formance of the bioelectrode in terms of electrocatalytic sensitivity
and operational stability. The highest catalytic peak current, well-
deﬁned even after 30 potential sweeps, was obtained with PGE
–Hb–CTAB electrode prepared in the presence of 0.02 mol L
CTAB (i.e., higher than its CMC
). The optimal molar ratio of sol
O) was selected as 1:24 as it led to the best
operational stability upon successive measurements while main-
taining an acceptable sensitivity, even if high sensitivities (but
lower long-term stabilities) were also observed in a wider compo-
sition range (Fig. 3). We assume that the reason of signal stabiliza-
tion at PGE SiO
–Hb–CTAB is the interaction of the protein
molecules with the micelles of CTAB that entrapped protein mole-
cules and provided more homogenous distribution of encapsulated
Hb in the silica ﬁlm on the PGE surface. As for PGE SiO
electrocatalytic currents sampled at PGE SiO
linearly with raising of Hb concentration in the synthesis sol from
up to 40
and reached saturation at an Hb con-
tent of 50
Integrating the surface area of the cathodic peak obtained in
anaerobic conditions enables to estimate the surface concentration
of electroactive Hb, which was equal to (3.5 ± 0.7) 10
. Taking into account the electrode surface area
) and the surface that would occupy a single protein
(5.5 7.0 nm
), the above value would correspond roughly to 7
monolayers, indicating that not only Hb species contacting directly
the electrode surface are responsible for the voltammetric signal.
From the dependence of cathodic peak potential on scan rates,
the apparent heterogeneous electron transfer rate constant was
estimated as k
= 0.6 s
using the method developed by Laviron
 for a surface-controlled electrochemical system. The obtained
results indicate that the electron transfer of Hb entrapped in the
–Hb–CTAB ﬁlm is a fast process. This k
value is slightly lower
than for a SiO
–Hb ﬁlm electrogenerated on glassy carbon
= 1.2 s
For the PGE SiO
–Hb–CTAB obtained under the optimal condi-
tions, the inﬂuence of scan rate and pH on the voltammetric char-
acteristics was investigated. Peak currents were found to be
directly proportional to the potential scan rate with linear regres-
sion equation I(
A) = 0.48 + 0.02 v (mV s
)(R= 0.99) in the
10–100 mV s
range, indicating a thin-layer electrochemical
behaviour. Peak potentials were strongly affected by pH, shifting
in the negative direction when decreasing acidity, with an average
slope of 120 mV/pH (Fig. 5, curve 1), which is higher than the ex-
pected value (56 mV/pH at t=18°C)  for a proton-coupled
single electron transfer process probably because of incomplete
reversibility of the system. Measuring peak currents as a function
of pH in oxygen free solution has revealed an optimal pH value
around 6, giving rise to highest signal intensities.
3.3. Interest of Au nanoparticles addition into the biocomposite
UV–vis spectroscopy was ﬁrst used to conﬁrm the presence of
the Au particle in silica sol. The absorption spectra of Au, Hb and
their mixture in aqueous solution, as well as in silica sol, are pre-
sented, respectively, in Fig. 6a and b. The maximum of adsorption
spectra of Au colloid observed at k= 520 nm (Fig. 6a, curve 2) is
typical for the Au particles with diameter 3–40 nm . Absorp-
tion spectra of Hb were essentially changed in the presence of Au
(Fig. 6a, curves 1 and 3). Two new maxima at 540 and 570 nm
can be attributed to the reduced form of Hb [47,48]. The red shift
of absorption maximum to 600 nm observed for Au in silica sol
(Fig. 6b, curve 2) indicates aggregation of the particles in the sol
[37,38]. No maximum at 600 nm was found in UV–vis absorption
Fig. 3. Effect of TEOS:H
o molar ratio in silica sol on the catalytic peak current after
2 (1), 10 (2) and 30 (3) potential sweeps for PGE SiO
–Hb–CTAB in the presence of
. Concentrations in silica sol: CTAB – 0.02 mol L
conditions as in Fig. 1.
Fig. 4. Dependence of catalytic peak currents on the number of potential sweeps at
–Hb (1), PGE SiO
–Hb–CTAB (2), PGE SiO
–Hb–Au (3) and PGE SiO
CTAB–Au (4). Other conditions as in Fig. 1.
Fig. 5. Variation of peak potentials of cathodic signal for PGE SiO
–Hb–CTAB (1) and
–Hb–CTAB–Au (2) as a function of pH in anaerobic conditions.
36 T. Rozhanchuk et al. /Journal of Electroanalytical Chemistry 625 (2009) 33–39
spectra of the triple mixture of Hb, Au and silica sol (Fig. 6b, curve
3). Instead, two absorption bands with small intensity at 550 and
580 nm were observed similar to Hb–Au mixture. It can be con-
cluded that Hb prevents aggregation of Au particles in silica sol
and particular formation of reduced form of Hb is possible in triple
Hb–Au–silica sol mixture.
Gold nanoparticles (Au-NPs) were thus added to Hb–silica sols
(with or without CTAB) and these media were applied to prepare
Au-NPs–containing biocomposite ﬁlms electrogenerated on PGE
(i.e., PGE SiO
–Hb–Au and PGE SiO
of the surface of these modiﬁed electrodes by AFM conﬁrms the
good dispersion of Au-NPs within the ﬁlms and the absence of
large aggregates as the morphology of the ﬁlms did not change sig-
niﬁcantly in the presence or absence of gold into the biocomposite.
Roughness, as given by the root mean square parameter, increased
slightly after deposition of the thin sol gel ﬁlms (2.3 nm on bare
electrode and 3.7 nm on SiO
–Hb–CTAB), but the introduction of
nanoparticle did affect it in a signiﬁcant way (4.6 nm for SiO
Hb–Au and 2.8 for SiO
–Hb–Au–CTAB). On the other hand, X-ray
photoelectron spectroscopy (XPS) did not reveal any signal for
Au, most probably because of too low Au-NPs content and/or their
coverage by an Hb layer, making Au invisible by the surface anal-
ysis XPS technique. The presence of gold in the ﬁlm electrodes
was better evidenced by SECM, via approach curves recorded using
ferrocene dimethanol as redox probe (Fig. 7). To this end, two ref-
erence supports (a conductive gold plate and an insulating plastic
substrate) were used to highlight the positive and negative feed-
backs, (respectively for Au and plastic) classically observed when
approaching an ultramicroelectrode tip to the surface of conduc-
tive and non-conductive supports . As shown, the curve ap-
proach to the bare carbon electrode (curve c in Fig. 7) was the
very close as that observed with the gold plate (curve b in Fig. 7)
because of the conductive nature of the carbon electrode. When
covered with a SiO
–Hb–CTAB ﬁlm, however, a negative feedback
was recorded due to the presence of an insulating layer on the car-
bon surface (curve d in Fig. 7), but it was less pronounced than on
plastic (curve a in Fig. 7), indicating that the biocomposite ﬁlm
probably remains porous to the probe. In the presence of Au-NPs,
the approach curves turned to positive feedbacks (see curves e
and f in Fig. 7), but remained lower than that recorded for the bare
electrode (compare to curves b and c in Fig. 7), the lower current
values being observed for the CTAB-containing ﬁlm (curve f in
Fig. 7), which can be explained by its more hydrophobic character.
Despite this last limitation, the introduction of nanoparticle into
the ﬁlms improves the reactivity of the interface.
–Hb–Au and PGE SiO
have been characterized by cyclic voltammetry in the presence of
oxygen and the results illustrated in Figs. 2 and 4 (curves 3 and
4) indicate clearly a beneﬁcial effect of Au-NPs on the electrocata-
lytic response of the PGE SiO
–Hb–CTAB–Au in terms of sensitivity
and long-term stability. The best electrocatalytic activity appears
in the form of lowering overpotentials for PGE SiO
–Hb–Au in com-
parison to PGE SiO
–Hb, which was even better in the presence of
CTAB. Even more interesting is the dramatic increase in operational
stability of the Au-NPs-containing bioelectrodes for which only a
decrease by about 20% was observed after 30 successive potential
sweeps (see Fig. 4, curves 3 and 4). The optimized sol–gel mixture
giving rise to the highest catalytic currents and the most stable re-
sponse to oxygen upon multiple successive measurements with
–Hb–CTAB–Au was the following one (components ex-
pressed in molar ratio): TEOS:Au:CTAB = 1:4.9 10
a Hb concentration of 40
For PGE SiO
–Hb–CTAB–Au the amount of electroactive Hb mol-
ecules on the electrode surface was evaluated from integration of
the surface area of the CV peak obtained in anaerobic conditions
as (3.8 ± 0.9) 10
and the apparent electron transfer
rate constant k
estimated to be 0.5 s
from the dependence of
cathodic peak potentials on scan rates. Peak currents sampled at
–Hb–CTAB–Au were found to be directly proportional to
Fig. 6. Absorption spectra of Hb (1), colloidal gold (2) and mixture Hb + Au (3) in
aqueous solutions (a) and in a silica sol (b). C
(a and b curve 2),
Hb:Au molar ratio – 1:1 (a and b); pH = 6.0.
Fig. 7. SECM approach curves recorded in a solution containing 0.1 mol L
1 mmol L
over a ﬂat plastic surface (a), a gold electrode surface (b),
the bare carbon electrode surface (a) and the same electrode covered with SiO
Hb–CTAB (b), SiO
–Hb–Au (e) and SiO
–Hb–CTAB–Au (f). Electrode speed was
T. Rozhanchuk et al. / Journal of Electroanalytical Chemistry 625 (2009) 33–39 37
the potential scan rate with linear regression equation I(
0.89 + 0.02
)(R= 0.99) in the 10–100 mV s
cating again a thin-layer behaviour. Finally, the effect of solution
pH on voltammetric signals obtained in anaerobic conditions was
less pronounced in the presence of Au-NPs, causing a negative shift
of potential values with the slope of 43 mV/pH, which is much
less than in the absence of gold (Fig. 5). Peak current depends on
pH for both PGE SiO
–Hb–Au and PGE SiO
pH range was 5.5–6.5 which is wider than for the electrodes mod-
iﬁed without addition of Au-NPs.
The main characteristics of the four bioelectrodes studied here
are summarized in Table 1. For all modiﬁed electrodes fast electron
transfer processes were taking place. Simultaneous addition of
CTAB and Au nanoparticles into silica sol caused the most positive
shift of E
, improved stability of electrocatalytic signals and in-
creased amount of electroactive proteins on the surface. Combina-
tion of all these advantages in a single device has led to choosing
–Hb–CTAB–Au for further investigations.
3.4. Application of PGE SiO
–Hb–CTAB–Au for determination
and antivirus drug
For PGE SiO
values were proportional to the
concentration of dissolved oxygen (Fig. 8a). In the O
range from 0.5 mg L
to 9.0 mg L
, the linear regression equation
A) = (0.09 ± 0.06) + (1.53 ± 0.02)c(mg L
) with a correlation coefﬁcient of 0.999. The detection
limit was found to be 0.12 mg L
(3S criteria). The concentration
of dissolved O
in a tap water determined by the present voltam-
metric method using PGE SiO
–Hb–CTAB–Au was 7.1 ± 2.0 mg L
= 0.11). This is in very good agreement with data obtained by
the standard Winkler titration method , which gave a value
equal to 6.8 ± 0.2 mg L
= 0.02), pointing out a satisfactory
behaviour of the electrochemical method described here (good
reproducibility and accuracy).
In the presence of antivirus drug amino derivative of adaman-
tine (rimantadine) in phosphate buffer solution (pH 6.0) at con-
stant amount of dissolved oxygen the catalytic reduction current
of dissolved O
noticeably decreased after 3 min of PGE SiO
CTAB–Au contact with the drug solution (Fig. 8b). The catalytic
activity of the modiﬁed electrode was not renewed in supporting
electrolyte solution. Non-catalytic current of PGE SiO
Au in solution contained rimantadine wasn’t changed. Obtained
data can be a result of the interaction of the drug’s molecule with
the active centre of Hb which caused inhibition of its electrocata-
lytic activity. Similar results we obtained for different type of
amines and phenol which were shown to be the inhibitors of
peroxidase [50,51]. The decrease of I
was proportional to the
concentration of rimantadine in solution in the range from
0.5 mg L
to 2.0 mg L
in phosphate buffer (pH 6.0). The amount
of dissolved oxygen in solution inﬂuenced the slope of the calibra-
tion graph. But in the presence of buffer and under constant atmo-
spheric pressure and temperature of the solution the linearity of
the graph was satisfactory. At O
content 8 mg L
regression equation being
A) = (0.45 ± 0.34) + (3.40 ± 0.30)c
), R= 0.985, where
: catalytic cur-
rents of dissolved O
in the absence and in the presence of riman-
tadine in solution, respectively). For 0.5 mg L
of rimantadine in
solution the relative standard deviation (S
) was 0.04. The detec-
tion limit for rimantadine using PGE SiO
0.3 mg L
Incorporation of CTAB in the silica sol enhanced electrocatalytic
activity, and to improved operational stability of PGE modiﬁed
Some characteristics of PGE modiﬁed with various silica ﬁlms containing Hb.
Type of electrode I
Signal loss/% (after 30 measurements) E
/V (versus Ag/AgCl) Optimal pH range cHb/
–Hb 8.6 100 0.3 6.0 50 2.8 ± 0.6 1.2 
–Hb–CTAB 11.9 60 0.15 6.0 50 3.5 ± 0.7 0.6
–Hb–Au 12.0 20 0.22 5.5–6.5 40 2.2 ± 0.6 –
–Hb–CTAB–Au 11.9 20 0.09 5.5–6.5 40 3.8 ± 0.9 0.5
– electrocatalytic current of dissolved oxygen; I
– maximum of cathodic current in anaerobic conditions; U
– surface concentration of electroactive Hb; k
heterogeneous electron transfer rate constant.
Fig. 8. Variation of the cathodic peak currents measured at PGE SiO
as a function of dissolved oxygen concentration (a) and in the presence of antivirus
drug rimantadine (amino derivative of adamantine) (b). Concentration: (a) O
0–4 (a–g), inset represents calibration curve for determination of dissolved
; (b) rimantadine/mg L
0–1.5 (a–d), CO
= 8 mg L
, inset represents calibration
curve for determination of rimantadine. Supporting electrolyte solution:
0.01 mol L
phosphate buffer (pH 6.0) and 0.01 mol L
38 T. Rozhanchuk et al. / Journal of Electroanalytical Chemistry 625 (2009) 33–39
with electrogenerated SiO
–Hb–CTAB ﬁlm in comparison to PGE
–Hb. Doping further the sol–gel-derived bioelectrodes with
Au-NPs expanded the pH working range for the modiﬁed PGE
and greatly improved the stability of the oxygen reduction cata-
lytic currents. Simultaneous addition of CTAB and Au nanoparticles
in a silica sol containing Hb proteins led to high amount of electro-
active Hb, fast electron transfer rates and excellent stability of elec-
trocatalytic currents of the modiﬁed PGE. The PGE modiﬁed with
–Hb–CTAB–Au ﬁlm can be used for detection of dissolved oxy-
gen in the mg L
concentration range. It can be also extended to
the determination of antivirus drugs based on amino derivatives
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