SENSITIVE ELECTROCHEMICAL METHOD DEVELOPMENT
For “in vivo” Measurement of ROS in Ethanol Induced Stress
Lívia Nagy, Tünde Angyal, Geza Nagy
Department of General and Physical Chemistry, University of Pécs, Ifjúság útja 6., 7624 Pécs, Hungary
Matsumoto Akiko
Department of Pharmaceutical Science, University of Colorado, Aurora, CO 80045, USA
Jan Pribyl, Petr Skladal
Department of Biochemistry, Masaryk University, Kotlářská 2, 61137 Brno, Czech Republic
Keywords: Alcohol induced oxidative stress, Reactive oxygen species (ROS), and Electrochemical detection.
Abstract: The role of reactive oxidizing species (ROS) is proved within numerous physiological processes, including
aging, signal transduction and some kind of immune functions. Nowadays ROS and oxidative stress gain
increasing attention in connection with a wide spectrum of diseases. In case of Asian people, the enzyme
taking part in the ethanol metabolism, the aldehyde dehydrogenase is absent or mutated, that can result in
liver tissue damage upon extensive alcohol consumption. Most of the ROS species are electrochemically
active therefore the applications of electrochemical methods are the most promising for in situ or in vivo
monitoring or quantification of them. In our work development and improvements of selective and sensitive
method for electrochemical detection of these molecules and radicals are attempted. We prepared ultra thin
size-exclusion layer by electropolymerization of m-phenylenediamine monomer on the surface of the Pt
working electrode to ensure its selectivity. We have worked out the optimal circumstances for the selective
layer preparation and tested its stability and function. In order to enhance the sensitivity of ROS detection a
new amperometric method, the periodically interrupted amperometry (PIA) was developed and applied.
With this approach we succeeded selective and sensitive detection of H
2
O
2
in vitro.
1 INTRODUCTION
The participation of reactive oxidizing species
(ROS) in numerous physiological processes,
including aging, signal transduction and some kind
of immune functions is proved and their role is
intensively investigated. The ROS induced oxidative
stress is gaining growing attention in health care
sciences owing to its involvements in development
of a wide spectrum of diseases, such as
dermatological, neuronal, immunological disorders.
For example, it is well known that metabolism of
ethanol causes oxidative stress in liver tissue.
Oxidative stress is generated through the various
pathways related to ethanol metabolism (e.g.,
ALDH, microsomal ethanol oxidizing system), thus
leading to hepatic disease. We have previously
reported that one of the genetic polymorphisms
affects oxidative stress caused by ethanol using
model animals (Matsumoto, 2007 and 2008).
However, in that studies the experimental animals
had to be sacrificed for the analysis. Obtaining
closer view and saving life of experimental animals
could be resulted by using a proper method for local
monitoring of ROS species. Therefore, we decided
to introduce into our studies new effective and
reliable methods that allow in vivo monitoring.
Following changes of local concentration of
ROS in different areas of living subjects has been a
challenge for decades in experimental life sciences.
Since the life time of these species is quite short,
local detection is badly needed for understanding
their roles in different processes. They are
electroactive. Therefore application of electrometric
359
Nagy L., Angyal T., Nagy G., Akiko M., Pribyl J. and Skladal P..
SENSITIVE ELECTROCHEMICAL METHOD DEVELOPMENT - For “in vivo” Measurement of ROS in Ethanol Induced Stress.
DOI: 10.5220/0003157303590362
In Proceedings of the International Conference on Biomedical Electronics and Devices (BIODEVICES-2011), pages 359-362
ISBN: 978-989-8425-37-9
Copyright
c
2011 SCITEPRESS (Science and Technology Publications, Lda.)
methods has been attempted in different times for
gaining information about different steps of
physiologic happenings.
Fortunately ROS species are small molecules,
some of them even are highly volatile. Therefore
size exclusion modifying layer or “gas dialysis”
membrane employed on the electrode surface can
dramatically improve the chance of selective
voltammetric or amperometric detection.
Recently dramatic selectivity improvements
have been achieved by employing electrochemically
prepared polymer layers (Nagy, 2002).
Up till now the performance of ROS measuring
microelectrodes were investigated mostly in vitro
conditions, however in vivo experiments were also
performed.
In order to increase sensitivity of detection with
coated amperometric electrodes the method of
periodically interrupted amperometry (PIA) has been
introduced (Nagy, 2006). It employs short train of
measurement electrode potential pulses separated by
longer, equal relaxation periods. This measuring
program allowing time for reloading of the diffusion
layer provides higher current signal and therefore
improved sensitivity as well as lower limit of
detection.
In this paper we shortly introduce our recent
results achieved working out a sensor and a method
applicable for ROS measurements. Molecule
modeling, in situ atomic force microscopy (AFM)
and quartz crystal microbalance (QCM) experiments
combined with controlled potential electrolysis
(Pribyl, 2010) were employed in developing the
selectivity providing polymer layer. That part of the
work will be also discussed.
2 EXPERIMENTAL
2.1 Instrumentation
2.1.1 Quartz Crystal Microbalance (QCM)
Standard gain oscillator (10 MHz basis) connected
to frequency counter was used for QCM
experiments. Data were collected by LabTOOLs
software (Petr Skládal). Gold covered QCM sensors
(ICMFG, USA) with optically polished surface were
used in all experiments.
2.1.2 Atomic Force Microscopy (AFM)
NTgra Vita (NT-MDT, Russia) equipped with a
large-scanner head was used for the AFM
experiments. HA-NC tips (NT-MDT, Russia) were
used in all cases. Typical settings: resonance
frequency of the tip in air ~100 kHz (dumped to
about 20 kHz in liquid). Scanning speed was 0.8 Hz.
2.1.3 Electrochemistry
AUTOLAB 12 electrochemical workstation
controlled with software of GPES version 4.9.009
for Windows (Eco Chem B.V., Netherlands) and
CHI type 760C (CH Instruments Inc. Austin, Texas
USA), electrochemical workstations were used in
voltammetric experiments.
The measuring programs were taken from the
standard working menu of the apparatus.
PalmSens (PalmSens, Netherland) instrument
driven by PalmLite software was used for
electrochemical procedures in case of QCM and
AFM studies.
2.1.4 Molecular Modeling
HyperChem Professional 7.52 (academic version)
chemical software served for estimation of charge
distribution in monomers involved, for guessing
their orientation on platinum surface as well as to
determine and draw the structure and spatial
configuration of the electropolymer.
2.2 Measuring Methods
Controlled potential electrolysis was applied for the
deposition of the size exclusion layer, with 0.6 V
constant potential. Studying the electropolymer
formation with QCM, the gold film on the crystal
served for working electrode, while silver-chloride
coated silver wire reference and stainless steel
auxiliary electrodes were used. The polymer
formation was carried out in 0.1 M KCl.
Chronoamperometric method was applied for
detection of H
2
O
2
detection. A three electrode cell
was used, where the Pt working electrode was
covered with selective layer. 1mm OD Pt and 1 mm
OD Ag served as counter and reference electrodes
respectively.
2.3 Chemicals and Reagents
All reagents were of analytical grade and used
without any purification. All solutions were made
with double distilled water. The pH 7.4
physiological phosphate buffer solution (PBS buffer)
was produced by the Pharmacy Institute of Medical
Faculty, University of Pécs. The hydrogen peroxide
BIODEVICES 2011 - International Conference on Biomedical Electronics and Devices
360
was purchased from Molar Chemicals (Hungary,
03650-203-340). The
m-phenylene- diamine (mdpa)
used
for the preparation of the size exclusion layer
(SEL) was obtained from Sigma (USA, A7030). The
bovine serum albumin was also Sigma product. The
30% hydrogen peroxide was diluted to 100 mmolL
-1
by distilled water and the concentration of the
diluted solution was determined by iodometric
titration. The solution was further diluted for the
amperometric electrode calibration.
3 RESULTS AND DISCUSSION
For ROS measurement H
2
O
2
was our model
compound. To receive the best structured SEL
detailed study was made.
3.1 Modeling the Layer
To obtain the thinnest, most effective SEL molecular
modeling work was made. First the fine charge
distribution of each atom of the monomer was
calculated. The high charge values (-0.38) of the
nitrogen atoms and the delocalized π bond of the
carbon ring determine the possible orientation of the
mpda monomer on the platinum electrode surface.
3.2 Deposition of Ultrathin SEL
Relatively high electrode potential is needed for
detecting through amperometric oxidation hydrogen
peroxide or other ROS species. The presence of
other electroactive species would present serious
interference, especially at high concentration.
Therefore the size exclusion layer essentially needed
for selective ROS detection.
The following experimental conditions resulted
in optimal SEL according to our studies:
Controlled potential electrolysis at 0.4V vs.
Ag/AgCl for 5s in 10 mmolL
-1
solution of m-
phenylenediamine prepared with pH=7.4 phosphate
buffer as solvent.
While working out of the procedure, we
examined the effectiveness of the SEL making
amperometric measurements in stirred buffer
solution at 0.65 polarization voltage.
The selectivity check was performed for each
freshly prepared SEL in our further studies and only
well performing electrode was used. The SEL was
accepted as good one if the current change was
smaller than 5 pA after ascorbic acid concentration
change from 0 to 0.21 mmolL-1. (The electrode
diameter was 1mm.)
3.3 Testing the Thickness of the SEL
As it is well known the quartz crystal microbalance
(QCM) detects the mass of the surface deposited
material through the frequency change of its
piezoelectric quartz crystal resonator.
Monitoring of the resonance frequency in the
real time allows to follow directly the kinetics of the
surface processes without need of other detectors.
Electropolymerization of mpda monomer in 10
mmolL-1 concentration solution, at constant
potential 400 mV was carried out. The curve in
Figure 1 shows, the change of the resonance
frequency of QCM gold plated sensor in time.
Amperometric polymerization measurement was
performed five times for 5 s, and once for 60 s.
The first deposition looks effective, changing the
frequency by 452 Hz, while the other deposition
steps are much smaller. The deposited mass of mpda
is about 375.16 ng which corresponds to a few
molecular layer thickness of monomer.
900
800
700
600
500
400
300
200
100
0
100
0 200 400 600 800 1000 1200 1400 1600
time(sec)
fr
rel
(Hz)
Figure 1: Monitoring the layer formation by quartz-crystal
micro balance (QCM). The first stair represents the
formation of the 30 nm thick layer in 5 s.
3.4 Topography of Ultrathin SEL
As it is well known, atomic Force Microscopy
(AFM) is a high-resolution scanning technique
allowing visualization of surface morphology and
mechanical properties in a sub nanometer scale. This
high-resolution imaging method was applied to
check the surface structure of the p-mpda layer
deposited. Scanning the surface mechanically with
well-defined, precise movements, the changes were
detected. The well formed column structured layer
could be observed that gives 30 nm average
thicknesses as can be seen on Figure 2.
SENSITIVE ELECTROCHEMICAL METHOD DEVELOPMENT - For "in vivo" Measurement of ROS in Ethanol
Induced Stress
361
Figure 2: The AFM topology of the ultrathin p-mpda
covered working electrode.
3.5 Cell Development for in vivo
Alcohol induced ROS measurements in vivo, are
planed. A new cannula type electrode cell has been
developed for
experiments in body fluids of
anesthetized experimental animals e.g. lack of
ALDH2, knockout mice. The electrochemical cell- -
incorporated micro sized working electrode has the
same sensitivity as an OD 1 mm disk electrode.
3.6 PIA Measurement
The H
2
O
2
is a relatively stable ROS. Its stability in
two different media was checked with the
amperometric H
2
O
2
sensor. In one case, the 5 cm
3
PBS buffer, containing 35 g L
1
bovine serum
albumin, (known free radical scavenger) was
pipetted into the measurement cell and the
amperometric current was recorded at 0.7V
electrode potential. The solution was kept under
intensively stirring. After steady reading was
obtained, 10 μL doses of 1 mmol L
-1
H
2
O
2
solution
were added. After each addition the current
increased and achieved steady value. The current
was potted against concentration in order to obtain
calibration curves. The calibration was performed by
chronoamperometric and PIA methods in PBS
buffer solution.
4 CONCLUSIONS
Sensitive and selective ultrathin size exclusion layer
covered electrode was developed for ROS
measurement. In combined application of AFM-
QCM-electrochemistry the electrodeposition of
poly-phenylenediamine layer was followed and
studied.
PIA method developed in our laboratory, has
substantial benefits when small concentration of
ROS species needed to be followed in presence of
other electroactive species.
Further experiments are in progress for
development of methods capable of following
concentration changes of ROS resulted by alcohol
induced oxidative stress in vivo, in body fluids of
anesthetized experimental animals.
ACKNOWLEDGEMENTS
This study was supported by the National Office for
Research and Technology (NKTH) CZ-17/2008 and
the Talented Student Award of Pécs University
2010, TÁMOP-4.1.1-08/1-2009-0009 of EU project.
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