American Journal of Analytical Chemistry, 2013, 4, 607-615
Published Online November 2013 (
Open Access AJAC
Amperometric Hydrogen Peroxide Biosensor Based on
Horseradish Peroxidase Entrapped in Titania Sol-Gel Film
on Screen-Printed Electrode
Reza E. Sabzi1,2, Fereshteh Rasouli1, Farshad Kheiri3
1Department of Chemistry, Faculty of Science, Urmia University, Urmia, Iran
2Institute of Biotechnology, Urmia University, Urmia, Iran
3Department of Chemical Engineering, Urmia University of Technology, Urmia, Iran
Received August 23, 2013; revised September 23, 2013; accepted October 15, 2013
Copyright © 2013 Reza E. Sabzi et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
We report the fabrication of disposable and flexible Screen-Printed Electrodes (SPEs). This new type of screen-printed
electrochemical platform consists of Ag nanoparticles (AgNPs) and graphite composite. For this purpose, silver
nanoparticles were first synthesized by a chemical reduction method. The morphology and structure of the AgNPs were
analyzed using a Scanning Electron Microscope (SEM) and UV-Visible spectroscopy. Graphite was chosen as the
working electrode material for the fabrication of a thick-film. The fabrication of a screen-printed hydrogen peroxide
biosensor consisting of three electrodes on a polyethylene terephthalate (PET) substrate was performed with a spraying
approach (working, counter and reference: enzyme electrode, graphite, pseudo reference: Ag/AgCl). This biosensor was
fabricated by immobilizing the peroxidase enzyme (HRP) in a Titania sol-gel membrane which was obtained through a
vapor deposition method. The biosensor had electrocatalytic activity in the reduction of H2O2 with linear dependence on
H2O2 concentration in the range of 105 to 103 M; the detection limit was 4.5 × 106 M.
Keywords: Screen-Printed Electrode; Ag Nanoparticle; Titania Sol-Gel; Biosensor
1. Introduction
Hydrogen peroxide is a reactive oxygen species [1] and
is the simplest peroxide which has a higher capability of
oxidation. This molecule is a by-product of several oxi-
dative-biological reactions which are the main factors of
diseases such as asthma, cancer, neurodegenerative dis-
orders, heart disease, etc. [2-7]. It acts as an important
mediator in environmental, pharmaceutical, clinical,
industry, food analyses and medicine production [8-10].
Its determination based on a simple, credible, precise,
fast and economical method is very important. In order to
assay hydrogen peroxide, several techniques have been
applied, including chemiluminesence, Voltammetry, spe-
ctrometry, fluorimetry, and an electrochemical biosensor
[11-15]. Most of these methods, however, had disad-
vantages, such as complexity, high cost, time con-
sumption, the need to use expensive reagents, and the
existence of interference [16,17], which act as hindrances
to the accurate determination of hydrogen peroxide.
Additionally, the validity of most of these methods may
be under question. Taking all the above-mentioned shor-
tages into consideration, it can be said that electro-
chemical biosensing methods which are based on enzyme
electrodes not only do not have the deficiencies of pre-
vious methods, but also enjoy advantages such as high
levels of sensibility, selectivity, fast analysis, low con-
sumption of reagents, low volume of analyte usage, and
easy design and application [18,19]. Recently, some in-
ventive techniques for the fabrication of sensors have
been proposed: thick-and thin-film technology, silicon
technology, etc. Among these, the equipment needed for
thick-film technology is the most convenient and
cheapest, and therefore, is the most applicable method
for sensor production. The thick-film technique can be
defined as the sediment of ink on a substrate surface with
their pattern and thickness controlled principally by
screen-printing [20]. Screen-printing, as a subset of thick-
film technology, is flexible and versatile and provides the
possibility of form and size selection. Screen-printed
electrodes (SPE) are miniaturized tools constructed by
printing multiple successive layers of different com-
pounds, such as conductive carbon ink and various metal
pastes, onto various substrates, such as alumina, ceramic,
polyvinyl chloride (PVC), gold, and iron [21,22]. Due to
their mechanical and electrochemical stability, these
electrodes are good alternatives for classical electrodes.
Their disposable feature also resolves the problem of
pollution caused by previous tests. Based on the previous
extensive studies, an essential principle of the develop-
ment of SPE thick-film biosensors is enzyme immobili-
zation [23,24]. Several methods for this have been inves-
tigated, for example, adsorption, cross linking, using sol-
gel matrixes, and entrapment with polymer [25-29]. Des-
pite these studies, there are many problems concerning
simplification of fabrication, correct maintenance of en-
zymes on the biosensor surface, maintaining its activity,
and ultimately increasing the life of the biosensors. Most
of these techniques can be used simply, but due to the
weak binding of enzyme to substrate, some enzymes
permeate from its surface. Moreover, covalent techniques
are boring and require several chemical steps [30]. On
the other hand, because of their unique advantages, inclu-
ding preparation at low temperatures, chemical inertness,
tunable porosity, low leakage of materials, and thermal
stability, sol-gel methods have been extensively deve-
loped [31,32]. Sol-gel methods in low temperatures and
through hydrolysis and poly-condensation of an appro-
priate precursor such as TiO2, SiO2 create a three-di-
mensional inorganic network [33,34]. Common features
of a sol-gel matrix are porosity, surface area, polarity,
and stability which are dependent on the process of hy-
drolysis progression and condensation [31]. Several me-
thods such as spin-coating, drop-coating, radio frequency
sputtering, and chemical vapor deposition (CVD) are
practical for forming an active layer on the membrane’s
surface [35]. Among these methods, CVD is a chemical
process during which the substrate is exposed, for a spe-
cific time, to the evaporation of one or several precursors.
In this situation, sediment is formed on the surface of the
substrate by the precursor or reacts to it. Recently, with
the use of a new vapor deposition process, Titania sol-gel
is produced [36-38]. This method is simple and an easier
control of the chemical composition of layers than other
methods. It is also immune to the shortcoming that is
created by the acidic catalyst and calcinations step need-
ed in the traditional Titania sol-gel process [39-41]. The
enzyme, as a biocatalyst, loses its ability without the
existence of water, and no response can be seen in this
condition. Then Titania sol-gel membranes maintain
water that is stored within the adjacent layers of enzyme
[42]. In this work, we attempted to report on a novel
platform based on flexible polyethylene terephthalate
(PET) substrate in order to design a high-performance
electrochemical screen-printed biosensor. Due to the
usage of a self-assembly technique and a flexible poly-
mer substrate, the cost of the biosensor is very com-
petitive. In this research a simple and controllable vapor
deposition method was used to prepare a novel horse-
radish peroxidase (HRP) that entrapped Titania sol-gel
film on the surface of graphite composite sprayed onto a
flexible surface of polyethylene terephthalate (PET)
substrate modified with silver nanoparticles (Ag NPs)-
poly (vinyl chloride) (PVC) nanocomposite as a silver
paste. In the mentioned process, graphite composite was
chosen due to its electrochemical properties, low back-
ground current, and wide potential window. Ag nano-
particles exhibit the highest electrical and thermal con-
ductivity. The conductivity of Ag nanoparticles is a very
important factor; therefore, in order to achieve good step
coverage and to decrease the resistance scale, it is neces-
sary to control the percentage ratio of Ag nanoparticles to
PVC. Based on the findings of recent studies, the
designed HRP/Titania sol-gel/graphite membrane sys-
tems provide a biocompatible support for enzyme mole-
cules to efficiently retain their good activity and an op-
portunity to construct a sensitive amperometric hydrogen
peroxide screen-printed biosensor.
2. Experiment
2.1. Reagents and Chemicals
Horseradish peroxidase (HRP) was obtained from Sigma
Corp. (USA), used without purification, tetra-n-butyl or-
thotitanate (TBT), hydrogen peroxide (30%); high purity
graphite powder, poly (vinyl chloride) and tetrahy-
drofuran (THF) were purchased from Merck. Back-
ground electrolytes for electrochemical experiments were
0.1 mol·L1 phosphate buffer solutions (PBS) containing
0.1 mol·L1 KCl. All other chemicals were of analytical
grade and were used without further purification. All
solutions were made up with distilled water.
2.2. Apparatus
The electrochemical measurements were performed at
room temperature in a conventional one-compartment
cell with a three-electrode system consisting of an en-
zyme electrode as the working electrode, a graphite as
the counter electrode, and an Ag/AgCl (0.1 M KCl) as
the pseudo-reference electrode. Cyclic voltammetric
experiments were carried out in a static electrochemical
cell at 25˚C, while amperometric experiments were carri-
ed out in a stirred cell with a successive addition of hy-
drogen peroxide solution to the cell by applying an opti-
mum potential to the working electrode. These mea-
surements were performed with a -Autolab Type II
Open Access AJAC
R. E. SABZI ET AL. 609
potentiostat (EcoChemie B.V, Ultrecht, The Netherlands)
controlled by the Autolab GPES software version 4.9. A
freeze dryer (FD-550, Tokyo Rikakikai Co., Ltd.; Tokyo,
Japan) was used to remove water from the resultant
2.3. Synthesis of Silver Nanoparticles
Ag nanoparticles are provided based on following me-
thod: Firstly 600 mL solution of 0.01 M NaBH4 and
tri-sodium citrate (C6H5O7Na3) was provided, Then this
solution was titrated with 200 mL solution of 0.01 M
AgNo3 while being stirred and temperature controlled.
After that, a yellow solution was obtained which appro-
ved the formation of silver nanoparticles. Finally it was
dried in a freeze dryer for about 24 h to remove the water
from the resultant nanoparticles.
2.4. Preparation of Screen-Printed Electrodes
Screen-printed electrodes consisted of three printed elec-
trodes: an Ag/AgCl pseudo-reference electrode and two
graphite electrodes acting as working and counter elec-
trodes. The size of each electrode set was 1.5 × 3 cm.
The procedures used for the construction of the screen-
printed electrodes are shown in Figure 1. In the primary
stage, the forms of the mentioned electrodes were de-
signed separately (in the beginning stage working elec-
trode and then pseudo-reference and counter electrodes)
by template on two PET. It is necessary to point out that
this was designed in order to prevent the deposition of
Titania sol-gel (used to immobilize the enzyme applied
in constructing the biosensor discussed in the following
Section 2.5) on the surface of the pseudo-reference and
counter electrodes. Then the dispersed solution of silver
nanoparticles and PVC (95:5%) in the THF solvent was
sprayed on the surface of the designed electrodes. This
constructed layer supplied the conductivity character of
our electrode system. In the next phase, a compound so-
lution of graphite and PVC (97:3%) was sprayed on the
part of the modified surface with silver nanoparticles
Figure 1. Schematic diagrams detailing the fabrication steps
of a screen-printed electrode.
in working and counter electrodes. In order to eliminate
the remaining solvent used in producing compounds, the
produced film was cooked about 30 minutes at a tem-
perature of 60˚C. The pseudo-reference electrode was
made by deposing the Cl ion via placing an electrode
modified with NPs in KCl solution and potential (0, 1) V
applied for 10 seconds. Finally after the enzyme was
immobilized on the working electrode’s surface (Section
2.5), the manufactured electrodes were applied to deter-
mine hydrogen peroxide as a three electrode system.
2.5. Preparation of Enzyme Electrode
The HRP enzyme solution was first obtained by dissolv-
ing 5 mg HRP in 5 mL 0.1 M PBS (pH 7.0). 10 µL. The
HRP solution was dropped onto the surface of the screen-
printed working electrode pretreated as previously ex-
plained in this study. The electrode was then suspended
vertically above tetra-n-butyl orthotitanate in a sealed
flask and kept at a constant temperature of 25˚C for 6 h.
This resulted in the absorption of saturate tetra-n-butyl
orthotitanate vapor at 25˚C by the enzyme solution and
the slow formation of a Titania sol-gel membrane throu-
gh the hydrolysis of tetra-n-butyl orthotitanate on the
surface, trapping the HRP in the membrane to produce an
HRP/Titania sol-gel modified electrode. Finally, the bio-
sensor was immersed in a pH 7.0 phosphate buffer and
kept at 4˚C overnight to remove the excess enzyme from
the electrode surface. The biosensor was stored in a pH
7.0 phosphate buffer at 4˚C when it was not being used.
3. Results and Discussion
3.1. Characterization of Silver Nanoparticles
Metal nanoparticles include free electrons, which give
surface plasmon resonance (SPR) absorption band, due
to the combined vibration of metal nanoparticle electrons
in resonance with light waves [43]. By increasing the
time period of the aqueous component, the intensity of
the absorption band increases and consequently the color
changes from colorless to reddish-yellow. These charac-
teristic color changes are caused by the excitation of the
surface plasmon resonance in the metal nanoparticles
(Figure 2). (Inset A) shows the UV-Vis spectra of the
synthesized Ag nanoparticle solution in the wavelength
range of 300 to 800 nm. A characteristic peak at 412 nm
is clearly observed, which is indicative of the formation
of Ag nanoparticles. SEM is a powerful tool that pro-
vides an image of surface details and produces the sig-
nals represent of information on the surface topography,
composition, and other properties [44]. The SEM image
of AgNPs is shown in Figure 2 (Inset B). It can be seen
from the image that the AgNPs tend to form clusters and
that the diameter of one single AgNPs ranges from 62 to
Open Access AJAC
Figure 2. UV-absorption spectra (inset A) and SEM image
(inset B) obtained for synthesis of AgNPs solution.
84 nm.
3.2. Optimization of Value of Ag Nanoparticles
and Graphite in Screen-Printed Electrodes
In order to fabricate screen-printed electrodes with high
conductivity while simultaneously remaining stable, di-
ffe-rent percentages of Ag nanoparticles, PVC, and gra-
phite were examined. Observations are presented in the
following sections. Silver nanoparticles and PVC were in
(80:20 wt%), (85:15 wt%), (95:5 wt%) and (98:2 wt%)
with levels of resistance of 130, 40, 7, and 6 cm2 res-
pectively. It should be said that despite the increased
conductivity caused by the increased Ag nanoparticles,
adherence in (98:2 wt%) was low; therefore, with regard
to the two limiting factors of conductivity and adherence,
(95:5 wt%) from Ag nanoparticles and PVC were se-
lected. Graphite and PVC solution was sprayed on a sur-
face modified with Ag nanoparticles with percentages of
(85:15 wt%), (95:5 wt%), (97:3 wt%), and (99:1 wt%),
that ultimately acquired resistant levels of 300, 60, 20
and 14 ·cm2 respectively. Because (99:1 wt%) had a
low level of adherence, (97:3 wt%) from graphite and
PVC were selected.
3.3. Study of Coductivity of Modified
Screen-Printed Electrodes by Cyclic
Potassium hexacyanoferrate (III) is mainly selected as a
model for characterizing electrochemical systems in
aqueous solutions [45]. A valuble and convenient tool to
monitor the characteristics of surface-modified electrodes
is the cyclic voltametry of ferro/ferricyanide redox cou-
ple [46]. CV was conducted in 5 mM Fe(CN)6
3/4 and
0.1 M KCl at 100 mV s1 for a surface modified with the
optimum percentage of AgNPs to PVC (95:5%) and
different percentages of graphite powder. Figure 3 com-
pares the current response at each stage of the fabrication
process. As shown in Figure 3, curve C, the fabricated
-0.8-0.6-0.4-0.200.2 0.40.6 0.8
E/V vs . Pseudo Ag/Agcl
Current / µ
Figure 3. Cyclic voltammograms of modified electrodes
with PVC and graphite. A: 3% PVC + 97% graphite B: 5%
PVC + 95% graphite C: 15% PVC + 85% graphite. All
results were obtained in a 5 mM Fe(CN)6
3/4 solution (PBS,
0.1 M, pH 7.0), with the supporting electrolyte KCl (0.1 M),
and optimum percentage of AgNPs to PVC (95:5%).
electrode with 85 wt.% graphite showed a very small
signal response towards the FeIII/FeII redox couple. PVC
produced an insulating layer on the electrode that acted
as a barrier to the electron transfer between FeIII/FeII and
the surface modified with AgNPs, decreasing the anodic
and cathodic currents, but the redox probe of Fe(CN)6
revealed a reversible cyclic voltammogram with 95 wt%
graphite (Figure 3, curve B). When spraying the 3%PVC
+ 97% graphite composite onto the PET surface modified
with AgNPs to PVC (95:5%), a remarkable increase in
current was observed (Figure 3, curve A). This was due
to the fact that the increase in percentage of graphite
markedly promoted the electron transfer of the analyte
and the electrode surface and hence increased the current
3.4. Electrocatalytic Behavior of HRP Titania
Sol-Gel/Graphite Biosensor
Figure 4, inset (A) (curves a-e) shows the cyclic voltam-
metric behavior of the biosensor in the absence of H2O2
and in the presence of 1 × 105 - 1 × 103 M hydrogen
peroxide in PBS solution at a scan rate of 20 mV s1. As
seen in Figure 4 (curve a), no peak current appeared at
the CV curve of the biosensor in the absence of H2O2.
Also shown in Figure 4 (curves b-d), the cathodic peak
current increased dramatically upon the addition of 1 ×
105 - 1 × 103 M concentration of H2O2 at the surface of
the biosensor, which can be ascribed to the electro-
chemical reaction of the immobilized HRP. Figure 4,
inset (B) show calibration curve of biosensor, as can be
seen The response was proportional to the concentration
of hydrogen peroxide in the ranges of 0.01 mmol·L1 to
1.00 mmol·L1, while the detection limit was estimated to
be 4.5 µmol·L1 at a signal to noise ratio of 3. Based on
experimental results, the catalytic current is mainly based
on the electron transfer between HRP and the electrode,
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R. E. SABZI ET AL. 611
-0.85 -0.65 -0.45-0.25 -0.050.15
I / A
y = 7.5494x + 6.0549
= 0.9966
C/ mM
I / A
Figure 4. Inset (A), Cyclic voltammograms of the HRP/
titania sol-gel/graphite biosensor without (a) and with 1 ×
105 (b), 1 × 104 (c) and 1 × 103 mol·L1 (d) H2O2. Inset (B),
Calibration curve in the range of 1105 to 1103 M of
H2O2 concentration. Scan rate 20 mV·s1 vs. pseudo Ag/
AgCl in a phosphate buffer pH 7.0, containing 0.1 M KCl.
and the graphite membrane acts as a bridge providing an
electrical contact or pathway for electron transfer be-
tween the immobilized HRP and the base electrode
(AgNPs/PVC nanocomposite). The following Equations
(1)-(3) explain the electrocatalytic reduction mechanism
of HRP toward H2O2. HRP, as an oxidative heme-
containing enzyme, cleaves to the O-O bond of hydrogen
peroxide to form a first intermediate (compound I),
which is an unstable two-equivalent oxidized form con-
taining an oxyferryl heme and a porphyrin cation radical,
Compound II is the second intermediate from the first
reduction of the porphyrin radical cation, which retains
the heme in the ferryl state [47,48].
 
22 2
 
Compound IFe=OeHCompound II
  (2)
Compound IIeHHRPFeHO
 
  (3)
3.5. Effect of Operational Conditions on the
Biosensor Response
Various operational conditions such as pH, operating po-
tential, and temperature can affect the biosensor response.
The peroxidases are pH-dependent enzymes and exhibit
their maximum activities at different pH values [49]. On
the other hand, the ability of amino acids presented at the
active sites of the enzyme to interact with the substrate
depends on their electrostatic state, which in turn
depends on the pH of the solution; thus optimization of
the working pH for the enzyme electrode is considered to
be important [50]. The ionization states of the charged
groups of enzymes that retained their ionizations from
the last solution to which they have been exposed are
influenced by the pH value of the last aqueous solution
[42]. The dependence of the biosensor response on pH of
the measurement solution was investigated. A range of
pH values between 4.0 to 9.0 was studied. Figure 5 de-
picts the response increase from pH 4.0 and the increase
of the buffer pH which led to a decrease in response,
indicating that the catalytic response was controlled by
the enzymatic activity. A decrease of enzymatic activity
may lead to the decrease of response at high pH since
strongly acidic and alkaline environments result in the
denaturation of HRP [48]. The optimum response was
achieved in pH 5. The acquired pH is correspondent with
the isoelectric point of peroxide enzyme, but to ensure a
higher sensitivity and stability of the biosensor, we chose
a 0.1 M PBS (pH 7.0, containing 0.1 M KCl) for the
determination of hydrogen peroxide, the optimum pH
value for living organisms. The effect of operating poten-
tial on the response and background current of the bio-
sensor was studied, and an optimum signal-to-noise (S/N)
ratio was obtained at 180 mV (vs. Ag/AgCl pseudo-
reference electrode), which was selected as the applied
potential for amperometric measurements. The electro-
catalytic activity of enzymes is strongly dependent on
temperature; hence the effect of temperature on the bio-
sensor response was studied. With an increase in tem-
perature, the response time decreased because of the in-
creased activity of the enzyme at higher temperatures. In
order to maintain the stability and reproducibility of the
biosensor for a long time, we thus chose room tem-
perature as the operating temperature in our experiments.
3.6. Amperometric Response of the Biosensor
In order to study the performance of the biosensor during
the H2O2 detection, current-time experiments were per-
formed. Figure 6 shows the biosensor’s current-time
response to successive step changes of H2O2 concentra-
tion under optimized experimental conditions. After sta-
bilization of the background current, 10 µL of H2O2 solu-
tion was successively added to the PBS (pH 7.0) buffer
solution. Uponthe addition of H2O2 to the stirring PBS
I /
Figure 5. Effect of pH on the performance of HRP/titania
sol-gel/graphite biosensor in 0.1 M phosphate buffer con-
taining 50 µM H2O2 at 25˚C and Eapp = 180 mV.
Open Access AJAC
2070120 170 220 270 320
Figure 6. Amperometric response of the fabricated biosen-
sor to successive addition of H2O2 in a stirred 0.1 M PBS
(pH 7.0) with an applied potential of 0.18 V vs. Pseudo
reference Ag/AgCl.
buffer solution, the biosensor indicated a rapid and sensi-
tive response. The linear response of the biosensor was in
the range of 105 to 103 M, with a detection limit of 4.5
× 106 M, and the acceptable response was obtained at
pH = 7.0. The current responses were reproducible over
the concentration range of 105 to 103 M (RSD = 4.6%,
n = 6).
In Table 1 a comparison of the biosensor developed in
this study with others based on HRP is shown. The com-
parison indicated that the porous structure of Titania sol-
gel matrix provides an acceptable detection limit, long
linearity, and high stability in comparison with other bio-
sensors that need very expensive materials. Considering
the other advantages of this biosensor, such as its simp-
licity of preparation, portability, and relatively low cost,
this type of biosensor can be potentially commer-
cia-lized for the detection of hydrogen peroxide.
4. Conclusion
This work developed a novel biosensor for hydrogen
peroxide based on screen-printed electrode technology
by immobilizing horseradish peroxidase (HRP) in Titania
sol-gel matrix using a vapor deposition method. The
suggested sol-gel method retains HRP biological activity
since it provides a mild immobilization process for en-
zyme and a biocompatible microenvironment around the
hydrogen peroxide. The porous structure of the Titania
sol-gel matrix is very efficient in preventing HRP
leakage out of the film, resulting in the enzyme’s good
loading, high catalytic activity and the biosensor’s fast
response rate. In contrast to official methods, simple
preparation and the short time of analysis are the main
advantages of this biosensor. The application developed
in the current study highlights the criterion of flexibility
Table 1. Comparison of the proposed H2O2 biosensor with
other biosensors based on HRP.
Immobilization matrix Linear
range (M)
limit (M) Reference
5 × 107 to
5.2 × 103 1 × 107 [51]
5 × 106 to
3.8 × 103 6 × 107 [52]
5 × 106 to
5.1 × 103 1.7 × 106[48]
5 × 107
to 1.5 × 103 3.75 × 108[19]
5 × 107 to
2.0 × 105 5 × 107 [18]
Con A/Au-HRP 5 × 106 to
1.2 × 102 2.9 × 106[53]
1 × 105 to
1 × 103 4.5 × 106this work
ATP: 4-aminothiophenol, CH: chitosan, ITO: indum tin oxide, GS: sulfo-
nated graphen, GNPs: gold nanoparticles, Thi: thionine, MG: methylen
green, Con A: concanavalin A.
resulting from coupling screen-printing technology with
the use of a simple compound to produce biosensors.
5. Acknowledgements
Special thanks to Mr, Fereydoon Rasouli for his support
and encouragement during this study.
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