Sensors and Actuators B 160 (2011) 1063–1069
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Sensors and Actuators B: Chemical
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AC-electrophoretic deposition of metalloenzymes: Catalase as a case study for
the sensitive and selective detection of H
2
O
2
Malika Ammam
∗
, Jan Fransaer
Department of Metallurgy and Materials Engineering (MTM), K.U Leuven, Kasteelpark Arenberg 44, B-3001 Heverlee, Belgium
a r t i c l e i n f o
Article history:
Received 21 July 2011
Received in revised form 8 September 2011
Accepted 9 September 2011
Available online 16 September 2011
Keywords:
AC-electrophoretic deposition
Metalloenzymes
Catalase
H2O2 biosensor
a b s t r a c t
The deposition of metalloenzymes on electrodes in their highly active state using electric fields is chal-
lenging because the presence of metal cations within the enzyme structure renders the enzyme more
fragile and high applied currents or voltages may pull out the metal cations from the enzyme structure
and lead to its denaturation. In this study, we demonstrate that catalase, a metalloenzyme with four por-
phyrin heme Fe(III) groups can be deposited using AC-EPD to yield highly active enzyme layers. Under
the optimal deposition conditions of 30 Hz, 160 V
p–p
and 30 min deposition time using an unbalanced
triangular waveform, the catalase enzyme electrode had a sensitivity for H
2
O
2
of 32.5 nA/M mm
2
at a
polarization potential of -0.1 V vs. Ag/AgCl. The sensor has a linear response up to 90 M H
2
O
2
, a fast
response time of 4 s, a detection limit below 1 M and a reasonable stability without employing stabilizers
or an outer polymer layer.
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
Hydrogen peroxide (H
2
O
2
) is an important chemical used in
many industrial processes e.g. food processing [1–3], clinical prac-
tice [4,5], textile and paper pulp bleaching [6,7] and phenolic
polymerization [8]. In addition, hydrogen peroxide is the by-
product of reactions catalyzed by a large number of oxidases [9,10].
Therefore, determination of the concentration of H
2
O
2
is of great
interests for industrial, clinical and biochemical applications. Var-
ious methods have been used for H
2
O
2
determination including
titrimetry [11,12], spectrophotometry [13,14], fluorometry [15,16],
and chemiluminescence [17,18]. Nevertheless, these methods can
suffer from various interferences and may also be time-consuming
and expensive. Electrochemical methods are considered advan-
tageous over other methods in terms of their fast response, low
cost, simple instrumentation, high sensitivity and possibility for
miniaturization. In the last decades, the immobilization of enzymes
on electrodes for the design of amperometric biosensors for H
2
O
2
determination has been an area of intense research. Several types
of enzymatic electrodes have been developed for H
2
O
2
, based on
immobilized enzymes such as catalase. In view of this, catalase has
been immobilized by crosslinking in polymers and gels [19–26],
∗
Corresponding author. Present address: Faculty of Science, University of Ontario
Institute of Technology, 2000 Simcoe Street North, Oshawa, ON L1H 7K4, Canada.
Tel.: +1 905 721 8668x3625; fax: +1 905 721 3304.
E-mail addresses: m78ammam@yahoo.fr, Malika.Ammam@uoit.ca
(M. Ammam).
on a collagen membrane [27], in a lipid film [28], in carbon paste
electrodes [29], in didodecyl-dimethylammonium bromide liquid
crystal film [30], on a gold electrode modified with single-walled
carbon nanotubes [31]. Although the purpose of all these deposi-
tion methods is to immobilize the enzyme in its highly active state
for the development of sensitive and selective H
2
O
2
biosensors, the
procedures of these immobilization methods are very complicated
and most of the time led to a moderate sensitivity and selectivity.
Previously, we described glucose, glutamate and lactose biosen-
sors based on the deposition of glucose oxidase, glutamate oxidase
and -galactosidase on platinum electrodes using asymmetrical
alternating current electrophoretic deposition (AC-EPD), and have
shown that this results in sensors with very interesting char-
acteristics [32–34]. We have demonstrated that, compared to
other electrochemical deposition methods, thick and highly active
enzyme layers can be deposited. In addition, compared to other
immobilization methods such as encapsulation in polymer or gel
matrices and other manual techniques, the most obvious advan-
tage of AC-EPD is the automated manufacturing process. This leads
on the one hand, to the ease of the manufacturing process and, on
the other hand, to a high reproducibility. Here, we wanted to inves-
tigate whether enzymes from the group of metalloenzymes such as
catalase can also be deposited in their highly active states to yield
highly active biosensors. Catalase is a common enzyme found in
nearly all living organisms that are exposed to oxygen, where it
catalyzes the decomposition of H
2
O
2
into water and oxygen [35].
Structurally, it is formed by a tetramer of four polypeptide chains,
each over 500 amino acids long [36] and it contains four porphyrin
heme Fe(III) groups that allow the enzyme to react with H
2
O
2
. The
0925-4005/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.snb.2011.09.027