Quaternized chitosan as support for the assembly of gold nanoparticles and glucose oxidase: Physicochemical characterization of the platform and evaluation of its biocatalytic activity

Article abstract of DOI:10.1016/j.electacta.2010.10.022

We report for the first time the use of quaternized chitosan (QCHI) for the immobilization of gold nanoparticles (NP) and glucose oxidase (GOD), the characterization of the resulting platform and its biocatalytic activity using glucose as substrate. The c

Full text of DOI:10.1016/j.electacta.2010.10.022

Electrochimica Acta 56 (2011) 1316–1322
Contents lists available at ScienceDirect
Electrochimica Acta
journal homepage: www.elsevier.com/locate/electacta
Quaternized chitosan as support for the assembly of gold nanoparticles and
glucose oxidase: Physicochemical characterization of the platform and
evaluation of its biocatalytic activity
a
b
c
a
a,∗
Maria V. Bracamonte , Soledad Bollo , Pierre Labbé , Gustavo A. Rivas , Nancy F. Ferreyra
a
INFIQC, Departamento de Fisicoquímica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Haya de la Torre s/n, 5000 Córdoba, Argentina
Laboratorio de Bioelectroquímica, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, P.O. Box 233, Santiago, Chile
Departement de Chimie Moléculaire, Université Joseph Fourier, UMR CNRS 5250, 38041 Grenoble Cedex 9, France
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 July 2010
Received in revised form 6 October 2010
Accepted 9 October 2010
Available online 21 October 2010
We report for the first time the use of quaternized chitosan (QCHI) for the immobilization of gold nanopar-
ticles (NP) and glucose oxidase (GOD), the characterization of the resulting platform and its biocatalytic
activity using glucose as substrate. The chemical substitution of chitosan has allowed us to work at phys-
iologic pH to build up self-assembled layers of QCHI-NP as platform for the enzyme immobilization. The
adsorption of GOD was analyzed by surface plasmon resonance (SPR) to compare the surface coverage
of GOD in absence and presence of the QCHI-NP platform. The results obtained with cyclic voltamme-
try and scanning electrochemical microscopy (SECM) revealed that the adsorption of NP improves the
conductivity of the structure and its electrochemical reactivity, facilitating the oxidation of the hydrogen
peroxide produced by GOD. The electrodes modified with NP present higher amperometric response
demonstrating the efficient transduction of the enzymatic activity in this structure.
Keywords:
Quaternized chitosan
Gold nanoparticles
Scanning electrochemical microscopy
Surface plasmon resonance
Glucose oxidase
© 2010 Elsevier Ltd. All rights reserved.
1
. Introduction
Gold nanoparticles (NP) have demonstrated to provide a suit-
a copolymer of 1–4 linked of N-acetyl-␤-glucosamine and ␤-
glucosamine units. The biocompatibility, biodegradability, and
bioactivity of CHI associated with desirable physical and mechan-
ical properties have made this polymer an interesting element to
develop biosensors [7,8].
CHI and gold nanoparticles have been employed in different
designs of electrodes to develop (bio)sensors. In this sense, elec-
trochemical deposition has been recently reported to prepare a
chitosan–gold nanoparticles nanocomposite for the construction
of non-enzymatic [9] and enzymatic glucose sensors [10–13].
A biosensor based on LbL assembly of CHI, NP and GOD at Pt
electrode has been reported [14]. GOD was also immobilized in self-
assembled structures with CHI and the film was used to reduce a
gold salt to yield metallic gold nanoparticles [15].
able microenvironment for the immobilization of biomolecules.
The characteristics of gold nanoparticles such as high surface-
to-volume ratio, high surface energy, and their efficiency as
electron-conducting pathways have been the reasons to facilitate
the electron transfer between redox proteins and electrode sur-
faces [1–3]. These properties have led to an intensive use of this
nanomaterial for the construction of electrochemical biosensors
with improved analytical performance compared to other designs
of biosensors [4,5].
Different methodologies have been used to build up structures
combining NP and biomolecules. Among them, the layer-by-layer
(
LbL) technique, based on the electrostatic interaction of poly-
Despite of the successful use of natural CHI in the design of
biosensors, the polymer present the inconvenience of its low sol-
ubility at physiological pH [16]. In order to improve CHI solubility,
we substituted a percentage of CHI amine functions by methyl
groups to obtain quaternized amines. This chemical modification
gives place to a polymer, named quaternized chitosan (QCHI), with
permanent positive charge at any pH and soluble even in phosphate
buffer [17].
electrolytes of opposite charge, have demonstrated to be highly
successful. It allows the rational design of multilayered nanos-
tructures in a reproducible way and the efficient immobilization
of biomolecules [6]. The selection of the polymer to connect NP
and biomolecules is crucial for the analytical performance of the
resulting architecture. The natural polycation chitosan (CHI) is
We report for the first time, the use of QCHI for the immobiliza-
tion of NP and GOD, the characterization of the resulting platform
and its biocatalytic activity using glucose as substrate The pro-
cedure for the preparation of the electrodes involves two steps:
∗ Corresponding author. Tel.: +54 351 4334169; fax: +54 351 4334188.
E-mail addresses: ferreyra@fcq.unc.edu.ar,
nancy ferreyra@yahoo.com (N.F. Ferreyra).
0
013-4686/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.electacta.2010.10.022

M.V. Bracamonte et al. / Electrochimica Acta 56 (2011) 1316–1322
1317
first, the LBL self-assembly of QCHI and NP at thiolated gold elec-
trodes as electrochemical platform; and second, the adsorption of
QCHI-GOD multilayers on this platform as biosensing element. We
present the electrochemical characterization of the QCHI-NP plat-
form, the analysis of GOD adsorption at the QCHI-NP electrode by
surface plasmon resonance (SPR), as well as the analysis of the
bioelectrocatalytic activity of the immobilized GOD by scanning
electrochemical microscopy (SECM) and amperometry.
Construction of QCHI-NP platform: The multilayer was obtained
by alternating immersion of the electrode in QCHI and NP solutions.
−
1
QCHI adsorption was performed by immersion in a 0.50 mg mL
−
2
−3
QCHI solution prepared in 5.00 × 10 mol dm phosphate buffer
pH 7.40 for 20 min. NP were adsorbed for 60 min from the com-
mercial solution. After each adsorption step, the surfaces were
copiously rinsed with 5.00 × 10 mol dm phosphate buffer pH
7.40. The resulting electrodes are indicated as Au/MPS/(QCHI-NP)n,
being n the number of QCHI-NP adsorption steps. The effect of
NP adsorption time was evaluated by varying the immersion time
of Au/MPS/QCHI electrodes in the NP solution between 15 and
−
2
−3
2
. Experimental
1
20 min.
Construction of QCHI-GOD multilayer: The adsorption of the
enzyme was carried out by alternating immersion of Au/MPS elec-
2
.1. Reagents
Gold nanoparticles (NP) citrate stabilized of 10 nm nominal
−
1
−1
trodes in 0.50 mg mL QCHI solution for 20 min and 1.0 mg mL
GOD solution for 30 min. Both solutions were prepared in
5
adsorption step, the electrodes were copiously rinsed with the
phosphate buffer solution. The resulting electrodes are indicated as
Au/MPS/(QCHI-GOD)m, being m the number of QCHI-GOD adsorp-
tion steps.
The same procedures were done for electrodes previ-
ously modified with the QCHI-NP platform, being indicated as
Au/MPS/(QCHI-NP)n/(QCHI-GOD)m.
diameter (Catalog number G-1527) and glucose oxidase (GOD)
Type X-S, Aspergillus niger, EC 1.1.3.4, 210,000 Units per gram of
solid, MW 160 kDa), were obtained from Sigma. Hydrogen peroxide
(
−
2
−3
.00 × 10 mol dm
phosphate buffer pH 7.40. After each
(
(
30% V/V aqueous solution) was from Baker. Ferrocenemethanol
FcOH) and 3-mercapto-1-propanesulfonic acid (MPS) were from
Aldrich. Chitosan (Pronova, Norway) with molecular weight of
1
−1
90,000 g mol was used for preparing the quaternized chitosan
(
QCHI) as described in reference [17]. The average molecular weight
−1
of the resulting QCHI was 52,750 g mol and the content of quat-
ernized amine 40.0 mol%. Other chemicals were reagent grade and
used without further purification. All solutions were prepared with
ultra-pure water (18 Mꢀ cm) from a Millipore MilliQ system.
2.4. Measuring procedures
2
.4.1. SECM
2.2. Equipment
Is a powerful tool that makes possible to obtain information
about chemical reactivity at interfaces, and kinetic data of het-
erogeneous reactions [18]. It is a scanning probe technique that
uses a positionable UME as probe [19]. We employed SECM to
evaluate the electrochemical reactivity of the modified electrodes
using feedback mode (FB), Scheme 1A. This method is based on the
measurement of the current produced at the UME, iT, when it is
brought close to the substrate in the presence of a redox mediator
Cyclic voltammetry (CV) and amperometry were performed
with an EPSILON potentiostat (Bioanalytical Systems Inc., USA).
Scanning Electrochemical Microscopy was performed with a CHI
9
00 (CH Instruments Inc., USA). A ca. 10 ␮m diameter homemade
carbon fiber electrode served as ultramicroelectrode (UME), while
gold disk electrodes (Au) of 3 mm diameter (CHI 101) were used as
substrate. A rotating disk electrode (RDE) of gold (2 mm of diame-
ter, Radiometer Analytical, model EDI101 with tip model 35T110)
was used in connection with a speed control unit (Radiometer Ana-
lytical, Model CTV101). In all the experiments a platinum wire and
Ag/AgCl, 3 M NaCl (BAS, Model RE-5B) were used as counter and ref-
erence electrodes, respectively. The experiments were performed
at room temperature.
SPR measurements were done with a single channel, AUTOLAB
SPRINGLE instrument (Eco Chemie, The Netherlands). The SPR sen-
sor disks (BK 7) were mounted on a hemicylindrical lens through
index-matching oil to form the base of a cuvette. Sample solutions
[
20,21]. The steady-state current of the UME positioned far from the
substrate is iT,∞ = 4naFDC. In diffusion-controlled positive feedback
case, iT > iT,∞, the substrate acts as a conductive surface producing
an additional flux of the redox mediator at the UME. On the con-
trary, in the negative feedback case, iT < iT,∞, the substrate acts as
an electrical insulator and hinders the flux of the redox mediator
at the UME surface [22].
−
4
−3
In our experiments, FcOH (5.0 × 10 mol dm solution) was
used as redox mediator. The UME and the substrate potentials were
held at 0.500 V and 0.000 V, respectively, to allow the feedback
takes place between both electrodes (see Scheme 1A). A series of
constant high images of the modified electrodes was obtained fol-
lowing the procedure described in [3]. The results are presented
in a dimensionless form normalizing the experimental feedback
current, iT, by the steady-state current iT,∞.
(
60 ␮L) were injected manually into the cuvette. The measure-
◦
ments were carried out under non-flow liquid conditions at 25 C.
2
.3. Modification of gold surfaces
SECM was also used to detect the enzymatic activity of
the immobilized GOD. The analysis was performed in substrate
generation-tip collection mode (SG-TC) (Scheme 1B). In this oper-
ation method, the UME is approached to the surface modified with
the enzyme. In the presence of the substrate the reaction takes place
and the enzymatic activity is monitored at the UME by applying
a potential appropriated to detect an electroactive product of the
enzymatic reaction [23]. In our experiments, the detection of GOD
activity was based on the measurement of the hydrogen peroxide
formed according to the following reaction (see Scheme 1B):
The cleaning procedure of gold electrodes included polishing
with 0.05 ␮m alumina for 6 min, careful sonication in deionized
water for 5 min, and immersion in “Piranha” solution (1:3 v/v
H O /98% H SO ) for 5 min, followed by a rinsing step with
2
2
2
4
ultrapure water. Caution: “Piranha” solution is very corrosive and
must be handled with care. The clean surfaces were stabilized by
−
1
cycling the potential between 0.200 V and 1.650 V at 10 V s
in
.50 mol dm sulfuric acid solution until obtaining a reproducible
response. Before each experiment, the surface was checked by CV
−3
0
−1
−3
at 0.100 V s in fresh 0.50 mol dm sulfuric acid solution.
The adsorption of MPS was performed by soaking the electrode
MPS solution prepared in
sulfuric acid solution, followed by a careful
rinsing with deionized water.
GOD
Glucose + O −→ Gluconolactone + H O
2
2
2
−2
−3
for 30 min in a 2.00 × 10 mol dm
−
3
−3
1
.6 × 10 mol dm
The UME was first positioned using FcOH, as described in refer-
ence [3]. The surface was carefully washed and the cell filled with

1
318
M.V. Bracamonte et al. / Electrochimica Acta 56 (2011) 1316–1322
A
UME E
T
= 0.500 V
B
UME E
T
= 0.700 V
FcOH→ FcO .+
O
2
H O
2
2
d
d
FcOH FcO .+
Glucose + O
2
H
2
O
2 + Gluconalactone
GOD
Modified Electrode
Modified Electrode
E
s
= 0.000 V
s
E = o. c. p.
Feedback Mode (FB)
Subtrate Generation-Tip Colection
Mode (SG-TC)
Scheme 1. SECM experimental setup employed for (A) Feedback and (B) SG-TC mode. ET and Es, correspond to UME and substrate applied potential, respectively, and d is
the distance between the UME and the substrate.
−
2
−3
5
.00 × 10 mol dm phosphate buffer solution pH 7.40. The UME
plays images obtained at bare gold (a), Au/MPS/QCHI (b) and
Au/MPS/(QCHI-NP)1 with NP adsorption times of 60 (c) and (d)
120 min. The image of the bare electrode, presents a typical sub-
strate conductive behavior, with homogeneous current values of
1.25 times iT,∞ [22]. After QCHI adsorption, the normalized current
decreases to 1.12 iT,∞ as average. This behavior is compatible with a
negative feedback between the UME and the substrate, indicating
that QCHI partially blocks the electrochemical response of FcOH.
This effect is associated with the electrostatic repulsion between
FcO. formed at the UME and the positive charges of QCHI at the
modified surface. Similar behavior has been previously reported
for the adsorption of CHI at glassy carbon electrodes [28]. When
NP are adsorbed, the normalized current increases to 1.19 iT,∞ and
was then polarized at 0.700 V and an image of 250 ␮m × 250 ␮m
−1
was recorded at 20 ␮m s to obtain the background current. After
returning the UME to the starting position (x = 0, y = 0), the buffer
−2
−3
solution was replaced by a 5.00 × 10 mol dm glucose solution
and a new scan was performed. When GOD is active, the hydrogen
peroxide produced by the enzymatic reaction at the electrode is
oxidized on the UME at the applied potential producing an increase
in the current.
+
2
.4.2. Surface plasmon resonance (SPR)
This technique makes possible to monitor in situ interfacial pro-
cesses such as adsorption/desorption of molecules with molecular
weight higher than 1 kDa [24–27]. In this work, SPR experi-
ments were carried out to evaluate the adsorption of GOD at
Au/MPS/(QCHI-NP)n or at Au/MPS electrodes. Gold disks where
modified with MPS, rinsed with deionized water and inserted into
the SPR cell. An aliquot of 60 ␮L of 5.00 × 10 mol dm phosphate
buffer pH 7.40 was added to the cell to obtain a baseline signal.
The buffer was drained using a peristaltic pump and replaced by
1
.31 iT,∞, for 60 and 120 min, respectively. In the last case, the nor-
malized current is higher than iT,∞ of the bare electrode (note that
in Fig. 1d the current color scale is different). This electrochemi-
cal behavior can be associated with an increase in the electroactive
area of the surface or with a higher charge transference constant
of the redox compound at the modified surface. To evaluate this
aspect, we analyzed the electrochemical response of FcOH by CV
and RDE.
−2
−3
−
1
6
0 ␮L of 0.50 mg mL
QCHI solution. The interaction was mon-
itored until obtaining a constant value of SPR angle. The system
was then washed several times with buffer solution and 60 ␮L of
phosphate buffer where placed in the cell to record a new baseline.
The adsorption of GOD was performed in a similar way by using
−1
Fig. 2 presents the cyclic voltammograms obtained at 0.100 V s
−4
−3
−3
in 5.0 × 10 mol dm FcOH (prepared in NaClO 0.500 mol dm
)
4
at bare gold electrode (a), and at gold electrodes after adsorp-
tion of MPS (b), QCHI (c), and NP for 60 (d), and 120 (e) min.
As expected, for FcOH, the voltammogram at bare gold elec-
trode indicates a diffusion controlled oxidation process with peak
potential separation (ꢁEp) of 64 mV and anodic peak current
−1
6
0 ␮L of 1.0 mg mL GOD solution. The variation of SPR angle, ꢁꢂ,
was determined as the difference between the corresponding base
lines values before and after the adsorption step. Similar experi-
ments were performed to determine the amount of GOD adsorbed
at Au/MPS/(QCHI-NP)n/(QCHI-GOD)m electrodes.
(Ipa) of 2.8 ␮A, in agreement with previous reports [3,29]. Upon
modification with MPS and QCHI, the voltammograms exhibit
a decrease in peak currents of 4% and 22% and a substan-
tial increase of ꢁEp up to 87 and 123 mV, respectively. These
results indicate that MPS and QCHI behave as a barrier for the
electrochemical process. Accordingly, the kinetics of FcOH oxida-
tion process is determined by the rate of the electron transfer
across the film [29]. After NP adsorption, there is a diminution
of ꢁEp and enhancement of Ipa. The corresponding values for
2
.4.3. Amperometric experiments
Amperometric experiments were carried out by applying the
desired potential, allowing the transient current to decay prior to
the addition of the analyte and monitoring the subsequent current.
3
. Results and discussion
ꢁ
Ep and Ipa are 67 and 60 mV and 3.15 and 3.20 ␮A for, 60
3
.1. Electrochemical characterization of Au/MPS/(QCHI-NP)1
and 120 min of adsorption, respectively. These effects indicate a
faster electron transfer kinetics due to the presence of NP. Simi-
platform
lar results were also obtained using [Fe(CN)6]3 and [Fe(CN)6]
−
4−
3.1.1. Analysis by SECM, CV and RDE
as redox probes (not shown), in agreement with previous reports
30,31].
Gold electrodes modified by self-assembling of MPS, QCHI,
[
and NP were characterized by SECM using FB mode. Fig. 1 dis-

M.V. Bracamonte et al. / Electrochimica Acta 56 (2011) 1316–1322
1319
Fig. 1. Surface-plot images of (a) Au, (b) Au/MPS/QCHI, and Au/MPS/(QCHI-NP)1 with (c) 60 and (e) 120 min of NP adsorption time. Numbers on the images correspond to
−
4
−3
−3
normalized current values. Experimental conditions: 5.0 × 10 mol dm FcOH, supporting electrolyte: 0.050 mol dm phosphate buffer pH 7.40, ET = 0.500 V and Es = 0.000 V
−1
vs. (Ag/AgCl/3 M NaCl)/V and 10 ␮m s UME scan rate. The results are presented in a dimensionless form normalizing the experimental feedback current, iT, by the steady-state
current iT,∞.
To evaluate the effect of NP in the electron transfer process,
we determine the charge transfer constant, k , of FcOH by RDE.
response of hydrogen peroxide, we evaluate the effect of NP on the
electrochemical reaction of this compound by CV at Au/MPS/QCHI
and Au/MPS/(QCHI-NP)1 with increasing NP adsorption time
(Fig. 3). The electrochemical response of hydrogen peroxide is com-
pletely blocked at Au/MPS/QCHI electrode (a), while the oxidation
current at 0.700 V, are 66.2, 85.1, 139.8, and 142.0 ␮A for 30, 90,
120, and 150 min of NP adsorption time, respectively. The adsorp-
tion of NP clearly enhances the hydrogen peroxide electrochemical
response, being this effect more important for the oxidation than
for the reduction process. No additional increase in the currents
was observed after 120 min of NP adsorption, in agreement with
our results observed for FcOH and ferrocyanide.
0
−
−2
3
−1
−1
The values obtained from Tafel plots are: 7.0 × 10 cm s
for
for
−4
−2
bare gold, and 8.3 × 10 ; 2.8 × 10 , and 2.9 × 10 cm s
Au/MPS/(QCHI-NP)1 with 15, 30, and 120 min of NP adsorption,
respectively. These values clearly show a significant improve-
ment of the electron transfer process as the NP surface coverage
increases.
3
.1.2. Electrochemical response to hydrogen peroxide
Since the reaction used to detect the activity of GOD incorpo-
rated in the multilayer system is based on the electrochemical
1
1
50
00
4
3
1
0
.5
.0
.5
.0
e
d
a
b
e
d
c
c
b
50
0
-
-
1.5
a
3.0
0
-
50
.000
0.100
0.200
0.300
0.400
0.500
0.000
0.200
0.400
0.600
0.800
E/V
E/V
Fig. 2. Cyclic voltammograms obtained at 0.100 V s 1 on: (a) Au (- - -), (b)
−
Fig. 3. Cyclic voltammograms obtained at 0.100 V s
Au/MPS/(QCHI-NP)1 electrodes with (b) 30, (c) 90, (d) 120 and (e) 150 min of
−1
on (a) Au/MPS/QCHI and
Au/MPS ), (c) Au/MPS/QCHI ), and Au/MPS/(QCHI-NP) with (d)
(
(
−
2
−3
6
5
0 (—), (e) 120 min (
) of NP adsorption time. Experimental conditions:
.0 × 10 mol dm FcOH solution. Supporting electrolyte 0.500 mol dm NaClO4.
NP adsorption time. Experimental conditions: 1.00 × 10 mol dm
H2O2 solu-
−
4
−3
−3
−3
tion, supporting electrolyte 0.050 mol dm phosphate buffer pH 7.40. Potential vs.
(Ag/AgCl/3 M NaCl)/V.
Potential vs. (Ag/AgCl/3 M NaCl)/V.

1
320
M.V. Bracamonte et al. / Electrochimica Acta 56 (2011) 1316–1322
2
1
1
000
500
000
GOD
A
bl
ΔθGOD
bl
QCHI
5
00
0
ΔθQCHI
bl
0
600
1200
1800
2400
3000
3600
Time /s
5
4
3
2
1
000
000
000
000
000
0
B
Fig. 5. Surface-plot images obtained in SG-TC mode at Au/MPS/(QCHI-GOD) , (plot
1
b
a and c), and Au/MPS/(QCHI-NP) /(QCHI-GOD) (plot b and d), electrodes. Images
1
1
−
3
c and d were obtained in 0.050 mol dm glucose solution while images a and b
were acquired in phosphate buffer pH 7.40. Experimental conditions: ET = 0.700 V vs.
−
1
(
Ag/AgCl/3 M NaCl) and Es at o.c.p. Image parameters: 250 ␮m × 250 ␮m, 20 ␮m s
UME scan rate.
layer. Form these values; it is possible to obtain the amount of GOD
adsorbed on both surfaces [27]. Table 1 presents ꢁꢂ and the surface
coverage of GOD (ꢃ GOD) for the adsorption of 1, 2 and 3 bilay-
ers of (QCHI-GOD) at Au/MPS and Au/MPS/(QCHI-NP)1 platforms.
To calculate ꢃ GOD we consider that 120 millidegree of SPR angle
a
−
2
displacement corresponds to 1 ng mm of adsorbed protein [27].
1
2
12
−2
Average values of 4.3 × 10 and 2.1 × 10 molecules cm for each
GOD adsorption step were obtained at Au/MPS and Au/MPS/(QCHI-
NP) , respectively. The lower ꢁꢂ and ꢃ
values obtained in the
1
GOD
CHI 1
GOD 1
CHI2
GOD 2
End layer
CHI 3
GOD 3
presence of NP indicate that QCHI-NP layer is less efficient for the
following QCHI and GOD adsorption. Therefore, a larger amount of
enzyme is adsorbed in the absence of nanoparticles.
Fig. 4. (A) Sensorgrams for QCHI and GOD adsorption at Au/MPS. The arrows indi-
cate the injection of QCHI or GOD after recording the base line in buffer solution.
(
B) ꢁꢂ values for the adsorption of GOD at Au/MPS/(QCHI-NP) (a), and Au/MPS (b)
3
3
.3. Evaluation of GOD enzymatic response
platforms.
.3.1. SECM in SG-TC mode
3.2. Evaluation of GOD adsorption by SPR
SECM experiments in SG-TC mode were carried out to evalu-
ate GOD activity at the modified surfaces. Fig. 5 displays SECM
We evaluate the amount of GOD immobilized at two platforms:
images of Au/MPS/(QCHI-GOD) (plots a and c), and Au/MPS/(QCHI-
1
Au/MPS and Au/MPS/(QCHI-NP) . Fig. 4A displays the sensorgram
NP) /(QCHI-GOD)1 (plots b and d) platforms. In the last case the
1
1
for the adsorption of QCHI and GOD at Au/MPS. As it was previously
selected NP adsorption time of NP was 60 min. The activity of the
enzyme confined to the electrode surface can be clearly observed
from the corresponding difference between the current values
before (a and b) and after (c and d) the addition of glucose. Aver-
age background currents of 2 and 5 pA are detected, while after the
addition of glucose an increase up to 15 pA and 30 pA is observed
for Au/MPS/(QCHI-GOD)1 and Au/MPS/(QCHI-NP) /(QCHI-GOD) ,
respectively. The adsorption of one bilayer of QCHI-NP as plat-
form for the enzyme adsorption makes possible to obtain a higher
enzymatic response. These results are associated to a higher elec-
trochemical reactivity of the surface due to the presence of NP,
−
3
indicated, the baseline was obtained in 0.050 mol dm phosphate
buffer solution pH 7.40. After the injection of QCHI, there is an
increase in the resonance angle shift, ꢁꢂ. After washing the sur-
face with phosphate buffer to remove the weakly bound QCHI, the
solution of GOD is injected and a new increase in the resonance
angle due to the enzyme immobilization is observed. The proce-
dure is repeated to obtain the structure with the desired number
of bilayers. Fig. 4B presents ꢁꢂ values for successive adsorption of
GOD and QCHI on Au/MPS/(QCHI-NP)1 (a), and Au/MPS (b) plat-
forms. For comparison, ꢁꢂ are normalized by the value of QCHI
1
1
Table 1
a
Shift of SPR angle (ꢁꢂ) and surface coverage (ꢃ GOD) for GOD adsorption as a function of the number of QCHI-GOD bilayers .
m
Au/MPS/(QCHI-GOD)m
ꢂ/102 millidegree
Au/MPS/(QCHI-NP)1/(QCHI-GOD)m
ꢁꢂ/102 millidegree
ꢁ
ꢃ GOD/1012 molecule cm−2
ꢃ GOD/1012 molecule cm−2
1
2
3
(12 ± 3)
(15 ± 3)
(14 ± 5)
3.8
4.7
4.4
(6.85 ± 0.09)
(7 ± 1)
2.1
2.2
1.9
(6 ± 2)
a
The values correspond to the average of three independent experiments.

M.V. Bracamonte et al. / Electrochimica Acta 56 (2011) 1316–1322
1321
Table 2
Analytical parameters for Au/MPS/(QCHI-NP)n/(QCHI-GOD)m electrodes in the presence of glucose.
1
−1
−3
−3
−3
−3
Structure
(Sensitivity/10 ) ␮A M
(LOQ/10 ) mol dm
(Linear range/10 ) mol dm
Time of responses
n
m
0
1
2
3
1
2
3
1
2
3
1
(4 ± 1)
1.77
0.42
0.20
0.88
0.24
0.09
0.39
0.16
0.22
0.39
0.5–1.5
0.1–1.5
0.06–2.15
0.3–1.3
0.07–1.6
0.03–1.0
0.12–1.1
0.08–2.0
0.07–1.6
0.1–1.0
4.0
4.5
5.0
5.0
4.5
4.5
2.0
3.0
2.3
2.0
(16 ± 3)
(23 ± 3)
(9 ± 1)
1
2
3
(25 ± 2)
(29 ± 5)
(18 ± 2)
(31 ± 3)
(26 ± 2)
(19 ± 5)
Multilayers were built on the top of Au/MPS electrode. Each value is the average of five independent experiments.
since SPR experiment showed that the amount of GOD adsorbed
on Au/MPS/(QCHI-NP)1 is lower than on Au/MPS/QCHI. Experi-
ments made in different regions of the same surface showed small
variation between current values, indicating a homogeneous dis-
tribution of the enzyme on the modified electrode.
as it is shown in Fig. 6A, that corresponds to the amperomet-
ric recordings at 0.700 V for Au/MPS/(QCHI-GOD)2 (gray line), and
Au/MPS/(QCHI-NP) /(QCHI-GOD) (black line), for successive addi-
2
3
2
−
−3
tions of 0.20 × 10 mol dm of glucose and Fig. 6B that displays
their corresponding calibration plots. It is evident that the presence
of NP improves the response of the bioelectrode towards glucose.
Compared to other bioelectrodes based on the electrochemical
deposition of NP on CHI, the sensitivity obtained with our more
efficient platform was superior to that reported in [10] and similar
to that obtained in [11], while the response times (time required
to reach the 95% steady state response) were shorter than the
informed in those references.
Compared to other bioelectrodes based on self-assembled
multilayers containing NP, the response highly depends on the
polyelecrolyte. In the case of natural CHI, self-assembled struc-
tures of six bilayers of CHI/GOD/NP at a Pt electrode modified
3.3.2. Electrochemical response to glucose
The effect of the amount of NP on the enzymatic response of
GOD was also evaluated from calibration curves obtained from
amperometric experiments performed at 0.700 V for successive
additions of glucose. The sensitivities achieved at Au/MPS/(QCHI-
NP) /(QCHI-GOD)1 electrodes as function of NP adsorption
1
1
−1
3
1
−1
3
3
time were (4 ± 1) × 10 ␮A mol dm , (4 ± 1) × 10 ␮A mol dm ,
1
−1
3
2
−1
(
(
9 ± 1) × 10 ␮A mol dm , (1.5 ± 0.2) × 10 ␮A mol dm , and
2 3
−1
1.5 ± 0.3) × 10 ␮A mol dm for 0, 30, 60, 90 and 120 min,
respectively. An enhancement in the sensitivity to glucose is
observed with the increase in adsorption time of NP up to 90 min,
in agreement with the increase of NP surface coverage described
above.
−
1
with polyacrilic acid gave a sensitivity of 555 ␮A M , that cor-
−
1
respond to 92.5 ␮A M per bilayer [14], similar to the value we
obtain at Au/(QCHI-NP)1/(QCHI-GOD)1, but less than the value we
determined with Au/(QCHI-NP)2/(QCHI-GOD)1. On the other hand,
despite of the amount of protein adsorbed on to the QCHI-NP plat-
form is smaller, the values of sensitivity we obtained for the same
number of QCHI-GOD are 2.25 and 1.55 higher that those reported
in [17] for one and two bilayers, respectively.
To determine the biocatalytic activity of the enzyme incor-
porated in the structure we evaluate the response of different
platform, Table 2 summarizes the analytical parameters of bioelec-
trodes with different architectures. As the number of QCHI-GOD
bilayers increases, an augment of sensitivity and a decrease in
LOQ is observed. The comparison between the results obtained
with Au/MPS/(QCHI-NP)n/(QCHI-GOD)m electrodes with equiva-
lent number of QCHI-GOD bilayers (m) for 0, 1, 2 or 3 bilayers of
QCHI-NP shows that, in the presence of NP, there are an increase
of sensitivity, and a decrease of LOQ and time of response. The
most significant effect is obtained with two bilayers of QCHI-NP,
Ascorbic acid (AA), uric acid (UA) and acetaminophen (AP) in
biological sample could be easily oxidized at low over potential,
and often interfere with the detection of glucose. Even when the
main goal of this work was the physical chemical characterization
of the bioelectrodes, in order to evaluate its potential analytical
application we use the permselective polymer Nafion (NF) as anti-
1
0
0
0
0
0
.0
.8
.6
.4
.2
.0
A
B
0
0
0
0
.8
.6
.4
.2
b
b
a
a
0.0
0.0
0
1
2
3
4
5
6
7
8
9
10
0.5
1.0
1.5
2.0
2.5
3.0
t / min
-3
-3
(
cglucose /10 )/moldm
−
4
−3
Fig. 6. (A) Amperometric response and (B) calibration plots for successive additions of 2.0 × 10 mol dm at Au/MPS/(QCHI-GOD)2 (a) and Au/MPS/(QCHI-NP)2/(QCHI-
−
3
GOD)2 (b) electrodes. Experimental conditions: supporting electrolyte 0.050 mol dm phosphate buffer solution pH 7.40 and working potential 0.700 V vs. (Ag/AgCl/3 M
NaCl)/V.

1
322
M.V. Bracamonte et al. / Electrochimica Acta 56 (2011) 1316–1322
interference layer. We adsorbed the polymer either as external
layer or incorporated between QCHI-NP and QCHI-GOD bilayers. In
the last case the interference of UA, AA and AP are largely decreased,
although the selectivity is still non acceptable for glucose quantifi-
cation in blood samples. Further work is necessary to do to evaluate
and compare different alternatives to resolve this inconvenient.
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