Abstract

This study focuses on the development and characterization of amperometric biosensors based on H2O2-
HRP (horseradish peroxidase) and Prussian Blue (PB) modified screen-printed electrodes (SPE) in order
to improve the phenol detection. The performance of the modified biosensor was tested using cyclic
voltammetry and amperometry techniques. HRP was immobilized on PB modified SPE by sol-gel
entrapment in TMOS/MTMOS polymer matrices. The amperometric detection of H2O2 was performed in
KCl buffer at a xxxV versus Ag/AgCl. In terms of biosensor characterization, the good sensitivity of
56.58μA/mM and tiny detection limit of 450 Μa/cm2 mM endorse the use of this biocompatible phenol
sensor in medical and industrial applications due to good long term stability and excellent sensitivity.

Introduction

The horseradish roots are a rich source of
peroxidase, a heme-containing enzyme that
utilizes hydrogen peroxide to oxide a wide
variety of organic and inorganic compounds.
Although the term horseradish peroxidase is
used generically, the root of the plant contains
a number of distinctive peroxidase isoenzymes
of which the C isoenzyme (HRP C) is the most
abundant. This has been the subject of much of
the published work on HRP, which comprises
many thousands of papers in the scientific
literature. Some major advances in
understanding of the structure and function of
HRP C have been achieved relatively recently
and were initiated by the successful production
of recombinant enzyme. Smith et al. discovered
in 1990 the long awaited three-dimensional
structure of HRP C by X-Ray crystallography.
Their work on this enzyme was continued by
Gajhede et al. and Berglund et al. in 1997 and
2002 respectively, regarding the high resolution
description of the intermediates in the catalytic
cycle of the enzyme. Many physiological roles
have been assigned to HRP isoenzymes
including lignification, indole-3-acetic acid
metabolism, cross linking of cell wall polymers,
suberin formation and resistance to infection.
HRP-C comprises of a single polypeptide of 308
amino acid residues according to Welinder
(1976). The N-terminal residue is blocked by
pyroglutamate and the C-terminal is
heterogeneous, with some molecules lacking
the terminal residue Ser308. There are 4
disulphide bridges between cysteine residues
and a buried salt bridge. Nine potential N-
glycosylation sites can be recognized in the
primary sequence from which eight are
occupied. His170Ala mutant undergoes heme
degradation when H2O2 is added. On calcium
loss, enzyme activity decreases by 40%.
The enzyme uses the molecular oxygen as a
natural electron acceptor, the hydrogen
peroxide produced in the reaction can be
determined electrochemically. Prussian blue
modified electrodes represent very attractive
detectors for hydrogen peroxide because they
work at an applied potential around 0v versus
Ag/AgCl with no other electrochemical
interferences. Prussian blue is the most
commonly used electrochemical mediator for
analytical applications and has found a wide use
in the biosensor development. It`s characteristic
of catalyzing the hydrogen peroxide reduction
has been applied in the construction of
oxidoreductase based biosensors for clinical,
environmental and food analysis. The study
steps followed in this paper are briefly
mentioned as : immobilization of PB on SPE,
characterization by amperometry and cyclic
voltammetry, spectrometric determination of
HRP enzyme activity, immobilization of HRP by
sol-gel(alcoxide/hydrolysis; condensation) and
last the optimization and characterization of the
biosensor.
Experimental
Apparatus and instruments
onto the reference and counter electrodes that
could increase the internal resistance of the
system. Regarding the pre-treatment of the
electrode before the modification, the
electrode was kept for 30 minutes in phosphate
buffer solution. After the successful deposition,
the electrodes were dried at 60oC overnight in
order to obtain an active and stable layer of PB.
The PB-modified electrodes were stored in a
dark, dry and room temperature environment.

An Autolab PGSTAT 101 was used for cyclic
voltammetry (CV) and amperometric
measurements. Screen-printed electrodes
(Dropsens DRP-C110) (www.dropsens.com)
with carbon working electrode, silver pseudo-
reference electrode and carbon counter
electrode were used for the experiments. All
potentials are reported vs. pseudo-reference
silver electrode. All experiments were
performed at room temperature. During the
amperometric measurements, the convective
transport at constant speed was provided using
a magnetic stirrer and a stirring bar.
The as prepared sensor was tested via cyclic
voltammetry in order to establish the working
potential and via amperometry in order to
measure the sensoristic performances such as
sensitivity, linear response range, specific
sensitivity, detection limit and response time. In
the amperometric measurement, the PBS was
used for base signal while the standard addition
of hydrogen peroxide were used to determine
the parameters.

Reagents

K3[Fe(CN)6], H2O2, FeCl3, pyrogallol were

supplied by Sigma. HRP from xxxxxxxx .
TMOS (tetramethoxysilane ) was supplied by
Fluka. Standard solutions of H2O2, PBS in water
were prepared just before use.

Preparation of screen-printed modified

electrodes.

The modification of screen-printed electrodes

with Prussian Blue was accomplished by direct
precipitation of 0.1M K3[Fe(CN)6] and 0.1M
FeCl3 solutions , prepared in 0.01M HCl
following the next equation:

3K4[Fe(CN)6]+4FeCl3→Fe4[Fe(CN)6]3↓+12KCl

A drop of this mixture was carefully placed onto

the working electrode via micropipette
deposition avoiding any precipitation of the PB