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

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

 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

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

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

100

0 200 400 600 800 1000 1200 1400 1600

time(sec)

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

is a relatively stable ROS. Its stability in

two different media was checked with the

amperometric H

sensor. In one case, the 5 cm

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

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.

REFERENCES

Matsumoto, A., Kawamoto, T., Mutoh, F., Isse, T.,

Oyama, T., Kitagawa, K., Nakayama, K., Ichiba,.

M., 2008. Pharmacogenetics & Genomics 18(10) 847-852

Matsumoto, A., Ichiba, M., Horita, M., Yamashita, Z.,

Takahashi, T., Isse, T., Oyama, T., Kawamoto, T.,

Tomokuni, K., 2007. Alcohol 41(1) 57-59.

Nagy, L., Nagy, G., Gyurcsányi, R. E., Lindner, E.,

Neuman, M. R., 2002. J. Biochem. and Biophys. Meth.

53 165-175

Nagy, L., Kálmán, N., Nagy, G., 2006. J. Biochem.

Biophys. Meth. 69 133

Pribyl, J., Jilek, M., Skladal, P., 2010. Nanoeasure2010

Krakow, Poland 3-4 June

Tamaskó, M., Nagy, L., Wittmann, I., Mikolas, E.,

Molnar, G. A., Nagy, G., 2007. Physiological

Measurement 28 1533-1542.

BIODEVICES 2011 - International Conference on Biomedical Electronics and Devices

362