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DNA modified carbon paste

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voltammetric study of the
interaction between DNA and
acridine orange

Article in Chemia analityczna · January 2004

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Chem. Anal. (Warsaw), 49, 467 (2004)

DNA Modified Carbon Paste Electrode Applied to the

Voltammetric Study of the Interaction Between DNA
and Acridine Orange

by I.Ch. Gherghi2, S.Th. Girousi1*,

A.N. Voulgaropoulos1 and R. Tzimou-Tsitouridou2

1
Analytical Chemistry Laboratory, Department of Chemistry
2
Analytical Chemistry Laboratory, Department of Chemical Engineering
Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece

Key words: acridine orange, adsorptive transfer stripping voltammetry, DNA, CPE,
intercalating dye

Acridine orange is a dye which intercalates within DNA. The interaction of acridine orange
with double stranded (ds), thermally denatured single stranded (ss) calf thymus DNA and
poly (G) was studied in solution as well as at electrode surface by means of adsorptive
transfer stripping voltammetry (AdSTV) using carbon paste electrode (CPE) in 0.2 mol L-1
sodium acetate (pH 5.0).

Oran¿ akrydynowy wykazuje zdolnoœæ wstawiania siê w cz¹steczkê DNA. Zbadano oddzia-
³ywania oran¿u akrydynowego z poli-G oraz z dwuniciow¹ spiral¹ DNA (ds) i jednoniciow¹
(ss) uzyskan¹ w wyniku termicznej denaturacji DNA dwuniciowego (DNA pochodzi³o
z cielêcej przysadki mózgowej). Badania prowadzono w roztworze oraz na powierzchni
elektroy metod¹ adsorpcyjnej woltamperometrii stripingowej na elektrodzie z pasty wêglowej
w roztworze octanu sodu 0.2 mol L-1 o pH 5.0.

* Corresponding author. E-mail: girousi@chem.auth.gr

468 I.Ch. Gherghi, S.Th. Girousi, A.N. Voulgaropoulos and R. Tzimou-Tsitouridou

Nucleic acids are biomolecules consisting of electroactive groups like nitroge-

nous bases that can be reduced and / or oxidized at various electrode materials such as
carbon paste, glassy carbon and mercury [1,2].
The oxidation and reduction signals obtained are strongly influenced by the DNA
structure and the sensitively responds to DNA strand breaking or DNA binding, thus
serving as a very promising perspective in the development of electrochemical DNA
biosensors [2–6]. Certain chemical compounds interact with DNA inhibiting in this
way nucleic acid synthesis. This interaction is being realized in four different ways:
intercalation, nonintercalative DNA binding, covalent binding to DNA, strand break-
ing interactions [7].
Chemical compounds representing such behavior against DNA have attracted the
interest of chemists as they may possess potentially toxic (eg. environmental pollut-
ants) or therapeutic properties (e.g. as anti-cancer agents). These compounds are hydra-
zines [8], aromatic amines [9], PCBs-aflatoxins [4] and anti-tumor drugs [10–16].
Certain compounds, known as intercalators, considerably affect the electrochemical
behavior of DNA as a result of specific binding with DNA strands under the process
known as the intercalation process. The intercalation process can be studied electro-
chemically. The electrochemical study is mainly based on the differences in the elec-
trochemical behaviors of the nucleic-acid-binding molecules in the absence, and the
presence, of double-stranded DNA, including the shifts of the formal potentials of the
redox couple caused by the intercalation of nucleic-acid-binding molecules into DNA
double helix- and the reduction of peak current resulting from the dramatic decrease
in the apparent diffusion coefficient of the nucleic-acid-binding molecule after asso-
ciation with DNA [13, 17].
Acridine orange (AO) was studied as a model intercalator compound in the present
work, its chemical structure is the following:

Acridine orange [18] is a dye which intercalates within DNA and this behaviour
was electrochemically studied employing adsorptive transfer stripping voltammetry
(AdSTV) with differential pulse mode (DP) using carbon paste electrode (CPE). Mainly
the influence of acridine orange on the oxidation peak of guanine (potential and peak
height) were studied in order to investigate the behavior of acridine orange with DNA.
 DNA modified carbon paste electrode 469

EXPERIMENTAL

Reagents

Double stranded calf thymus DNA (Catalog No.D–1501, highly polymerized), poly–G (Catalog
No. P4404) was purchased from Sigma. Acridine orange hydrochloride (31,833–7) was purchased from
Aldrich and was of 99% p.a. The supporting electrolyte of differential pulse voltammetric experiments was
acetate buffer solution 0.2 mol L-1 (pH 5.0).
Thermally denatured single stranded DNA was prepared by boiling a solution of double stranded DNA
for 15 min and leaving it at 4°C for 10 min. The stock solution of dsDNA (1 g L-1) was prepared with
a solution of 10 mmol L-1 Tris-HCl and 1 mmol L-1 EDTA at pH 8.0. Stock solutions of acridine orange
hydrochloride hydrate were prepared with water. The dilute solutions were prepared just before use. The
water used was doubly distilled and sterilized.

Apparatus

Differential pulse voltammetric measurements were performed with a Metrohm 647 VA–Stand con-
trolled by a 646 VA–Processor. The working electrode was a carbon paste electrode of 6 mm diameter. The
reference electrode was a saturated Ag/AgCl and the counter electrode was a platinum wire. The carbon
paste was prepared in the usual way by hand-mixing graphite powder and nujol oil. The ratio of graphite
powder to nujol oil was 75:25. The resulting paste was packed tightly into a Teflon sleeve. Electrical contact
was established with stainless steel screw. The surface was polished to a smooth finish before use. The
electrode was pretreated by applying the potential of +1.7 V for 1 min without stirring prior to the accu-
mulation step. As it was already presented [19] the electrochemical pretreatment produces a more hydro-
philic surface state and leads to the concomitant removal of organic layers.
All water and pipette tips were sterilized by autoclaving for 20 min. The electrochemical cells were
cleaned with diluted nitric acid, rinsed with water and sterilized for 20 min. Ultrapure nitrogen was used to
deaerate the solutions for 5 min before each experiment.

Procedures

Interaction of solution-phase DNA with AO. The analysis of solution-phase DNA with AO consisted
of mixing the two components, followed by accumulation and transduction by transfer voltammetry with the
differential pulse mode. The electrode was rinsed with water for 5 s prior to each medium exchange. Stock
DNA (1g L-1) and AO solutions were added to 0.2 mmol L-1 acetate buffer to produce the required concen-
trations and the mixture was left to stand for 25 min. A freshly polished carbon paste electrode was im-
mersed into the mixture solution and was pretreated as described above. The accumulation of the mixture
was performed by applying a potential of +0.5 V for 2 min. The transduction was carried out in the blank
acetate buffer solution, with an initial potential of +0.1 V and a scan rate of 50 mV s-1.

Interaction of surface-confined DNA with AO. The procedure consists of DNA immobilization,
interaction of AO with immobilized DNA and transduction by transfer voltammetry with differential pulse
mode. Prior to each medium exchange, the electrode was rinsed carefully with water for 5 s. After the
pretreatment of the electrode, the nucleic acid was subsequently immobilized onto the electrode surface by
adsorptive accumulation for 2 min at +0.5 V. The dsDNA-coated electrode was transferred to the stirred
sample solution (analyte plus 0.2 mol L-1 acetate buffer solution pH 5.0) for 120 s, while holding the poten-
470 I.Ch. Gherghi, S.Th. Girousi, A.N. Voulgaropoulos and R. Tzimou-Tsitouridou

tial of +0.2 V. The transduction was performed in the blank acetate buffer solution. The same procedure was
followed for the immobilisation of ssDNA and the study of the ssDNA-sensor interaction with AO.

Interaction of surface-confined analyte with DNA. AO was first accumulated onto the surface of the
CPE and then the resulting acridine orange-coated electrode was immersed into the stirred sample solution
containing DNA plus 0.2 mol L-1 acetate buffer solution of pH 5.0. The surface of the electrode was renewed
prior to each assay. All the experiments were conducted at room temperature (20°C).

RESULTS AND DISCUSSION

Transfer voltammetry with differential pulse mode of AO, DNA and poly(G)
at carbon paste electrodes
In our studies we used 0.2 mol L-1 sodium acetate (pH 5.0) as a background elec-
trolyte which was more suitable compared with the 0.01 mol L-1 acetate buffer at pH
4.8 that was also examined. Native double-stranded (ds) DNA yielded a positive peak
at +0.99 V, which was more intense for the first background electrolyte. The same
happened with thermally denatured (single-stranded) DNA, which yielded a higher
peak at +0.989 V. In both of these two cases, the anodic peak corresponds to the
oxidation of the guanine residues. The synthetic polyribonucleotide poly (G) was
oxidized under the same experimental conditions and yielded a well-defined peak at
+0.994 V, as expected from its high guanine content.
It has to be mentioned that the concentration of DNA was optimized and found to
be 0.1 g L-1 for both dsDNA and ssDNA, in order to secure the full coverage of the
electrode surface [20].
The accumulation potential and the accumulation time have a profound effect
upon the response. Figure 1 indicates the effect of accumulation potential on the
oxidation signal of dsDNA for an accumulation time of 120 s. The peak current is
slightly affected by increasing the potential in the range 0.0 to + 0.6 V, while it de-
creased rapidly at higher potentials.
Acridine orange hydrochloride produces a well-developed peak at +0.78 V with
a preconcentration step at +0.2 V for 120 s. The response decays at other precon-
centration potentials, such as +0.4 V, +0.6 V and +0.7 V. The calibration curve at
Figure 2 shows the current response in relation to increasing concentrations of AO
under the above optimal conditions. Linearity is observed in the range 0–0.54 × 10-6 mol
L-1 (sens. 715.73 nA/µM, y = 715.73 x + 3.2336), while saturation of the electrode is
observed at higher concentrations.
 DNA modified carbon paste electrode 471

520

420

320
I, nA

220

120

20
0 300 600 900 1200
V, m V

Figure 1. Effect of accumulation potential on the differential pulse response of 0.1 g L-1 dsDNA; accumu-
lation was performed for 2 min

1000
500

400 1
900

300

V, mV
I, nA

200 800
2

100
700

-100 600
0 2 4 6 8 10 12
-7 -1
C[AO] ( x10 mol.L )

Figure 2. Calibration curve of AO. The CPE was freshly polished and immersed into the buffer solution
(0.2 mol L-1 acetate, pH 5) containing increasing concentrations of AO. The accumulation was
performed at +0.2 V for 2 min and the transduction was performed to blank acetate buffer.
1. Dependence of peak current of AO oxidation on concentration; 2. Dependence of the oxidation
potential on concentration of AO
472 I.Ch. Gherghi, S.Th. Girousi, A.N. Voulgaropoulos and R. Tzimou-Tsitouridou

Interactions of surface-confined DNA with AO in solution

The DNA-modified electrode was prepared by immersing the CPE into a solution
of dsDNA of the concentration 0.1 g L-1 in 0.2 mol L-1 acetate buffer (pH 5.0) for
2 min at +0.5 V. The electrode was washed and immersed in acridine orange solutions
of different concentrations ranging from 0 to 74 × 10-10 mol L-1 (in 0.2 mol L-1 sodium
acetate, pH 5.0, for 2 min at + 0.2 V). Differential pulse voltammograms were taken
after the transfer of the electrode into the blank background electrolyte. By increasing
the dye’s concentration, an increase in the DNA peak was observed, while the peak
potential of DNA shifted to more positive values. The DNA peak reached the maxi-
mum value when acridine’s concentration was 24 × 10-10 mol L-1. At higher concentra-
tions, the DNA peak started to decrease and finally leveled off. Figure 3 shows the
current changes of the characteristic guanine peak of dsDNA in relation to the acri-
dine orange concentration added. It has to be highlighted that when the AO concen-
tration reaches the value of 1.24 × 10-8 mol L-1 a new peak appears at +0.87 V which
is probably due to the formation of the dsDNA-acridine orange complex.

800 2
1
700
2
600

500

400
I, nA

300

200

100

0 200 400 600 800 1000

-10 -1
C[AO] ( x10 mol.L )

Figure 3. 1. Dependence of peak current of oxidation of guanine residues in dsDNA immobilized on the
electrode surface on concentrations of AO. The CPE was pretreated at +1.7 V for 1 min followed
by adsorptive accumulation of dsDNA at +0.5 V for 2 min and was immersed in AO solutions of
different concentrations; the incubation time prior to each scan was 2 min at + 0.2 V; 2. Depen-
dence of peak current of dsDNA-acridine orange complex on concentration of AO in the solution
 DNA modified carbon paste electrode 473

According to the previous studies [21] the positive shifts in Ep of metal com-
plexes are a result of their intercalation in dsDNA. In contrast to the intercalation
involving the hydrophobic interactions in the DNA double helix, electrostatic inter-
actions at the surface of the DNA molecule may result in a negative shift in Ep of the
interacting compound. So, we can interpret the appearance of the above peak as a sign
of the AO intercalation in DNA.

2
700
1
600 2
500

400
I, nA

300

200

100

-100
0 500 1000 1500 2000 2500
-10 -1
C[AO] ( x10 mol.L )
Figure 4. 1. Dependence of peak current of oxidation of guanine residues in ssDNA immobilized on the
electrode surface on concentration of AO; the CPE was pretreated at +1.7 V for 1 min followed
by adsorptive accumulation of ssDNA at +0.5 V for 2 min and was immersed in AO solutions;
the incubation time prior to each scan was 2 min at +0.2 V. 2. Dependence of peak current of
ssDNA-acridine orange complex on concentration of AO under the above conditions

Another impact of the binding of AO to dsDNA is the increase of the characteris-

tic oxidation peak of dsDNA with increasing concentrations of AO in the bulk solu-
tion, which is probably due to a bending of the DNA molecule and its ability to adhere
to the rough CPE surface [16]. In the case of the interaction of AO with dsDNA
immobilized onto the CPE, the dye binds preferentially to the sites of the duplex
oriented towards the solution. We have to take into account that DNA is electrostati-
cally adsorbed on the CPE surface through the negatively charged sugar-phosphate
backbone and forms a flat layer on the electrode surface. Also the side of the duplex
which is in the intimate contact with the electrode surface cannot be easily accessible
474 I.Ch. Gherghi, S.Th. Girousi, A.N. Voulgaropoulos and R. Tzimou-Tsitouridou

[22,23]. At low concentrations of AO the peak of DNA is increased because either

the molecules of AO are maybe hidden in the interior of the duplex or they are bound
to the side of DNA opposite to its contact with the electrode surface [16].
Such an increase was not observed in the case of thermally denatured (thermally
denatured single stranded) DNA. Figure 4 shows the current decrease of the charac-
teristic guanine peak in relation to acridine’s orange concentration. It has to be men-
tioned that when the acridine concentration reaches 3 × 10-8 mol L-1, a new peak
appears at +0.86 V, which could be attributed to the electrostatic binding between
ssDNA and AO.
It is obvious that in this case we cannot speak about an intercalative complex
formed as a result of the interaction between ssDNA and AO, but another mode of
DNA binding should be taken into account, such as the outer-sphere electrostatic
binding. The different result of the interaction at the CPE surface can be used as
a criterion for the existence of the intercalation effect.

Interactions of AO and DNA in solution

The incubation time of the two components is a very important factor affecting
the response.

800 1100

2
1000

600
900

800
V, mV

1
I, nA

400
700

600
200

500

0 400
0 10 20 30 40 50
Time of incubation, min
Figure 5. 1. Dependence of peak current of oxidation of guanine residues in dsDNA being in solution with
a constant concentration of AO (2 × 10-7 mol L-1) on the incubation time in relation to potential;
2. Dependence of the oxidation potential on the incubation time under the above conditions
 DNA modified carbon paste electrode 475

Figure 5 shows the effect of the incubation time of AO and DNA in solution. It is
shown that the current response is increased as the incubation time is increased. The
time selected was 25 min, since no dramatic change at the peak current occurs after
this point.
Double stranded DNA solution of 0.1 g L-1 was left to react for 25 min with
different concentrations of AO ranging from 0 to 98 × 10-8 mol L-1. Up to the concen-
tration 44 × 10-8 mol L-1, a subsequent increase in the DNA peak was observed. Then
the peak current of DNA leveled off, by increasing amounts of AO. Figure 6 shows
the dependence of the characteristic peak of DNA on the increasing amounts of AO
in the solution. It has to be mentioned that when the concentration of AO reached the
value of 44 × 10-8 mol L-1 a new peak started to appear at +0.86 V. This peak increased
with increasing the concentration of AO. We may thus conclude that there is a com-
plex formed in the solution and that the behavior is similar to the above case. In
Figure 7 a voltammogram of dsDNA alone as well as after the addition of two repre-
sentative acridine’s orange concentrations is observed.

900 2

800
1
700

600

500
I, nA

400

300

200

100 2
0

0 20 40 60 80 100
-8 -1
C[AO] ( x10 mol.L )

Figure 6. 1. Dependence of peak current of oxidation of guanine residues in dsDNA on concentration of

AO after incubation of stock dsDNA with AO to 0.2 mol L-1 acetate buffer for 25 min; the CPE
was immersed into the mixture solution, the accumulation was performed at +1.7 V for 1 min
followed by applying a potential of +0.5 V for 2 min; 2. Dependence of the characteristic peak
current of the dsDNA-acridine orange complex on concentration of AO into the solution under
the above conditions
476 I.Ch. Gherghi, S.Th. Girousi, A.N. Voulgaropoulos and R. Tzimou-Tsitouridou

10.00

7.65
I, µA

5.30

2.95

0.60
0.1 0.3 0.5 0.7 0.9 1.1 1.3 u, V

Figure 7. 1. Voltammogram of dsDNA (0.1 g L-1) in solution; 2. Voltammogram of the mixture dsDNA
(0.1 g L-1) + 44 × 10-8 mol L-1 AO in solution; 3. Voltammogram of the mixture dsDNA (0.1 g L-1)
+ 98 × 10-8 mol L-1 AO in solution

2
700
1
600

500

400
I, nA

300

200

2
100

0 20 40 60 80 100 120 140

-8 -1
C[AO] ( x10 mol.L )

Figure 8. 1. Dependence of peak current of oxidation of guanine residues in ssDNA on concentration of AO

after incubation of stock ssDNA solution with AO to 0.2 mol L-1 acetate buffer pH 5 for 25 min;
the CPE was immersed into the mixture solution, the accumulation was performed by applying
a potential at +1.7 V for 1 min followed by applying a potential at +0.5 V for 2 min; 2. Depen-
dence of peak current of ssDNA-acridine orange complex on increasing concentrations of AO
into the solution under the above conditions
 DNA modified carbon paste electrode 477

The same experimental procedure was performed using thermally denatured single
stranded DNA. Figure 8 shows a slight decrease in the peak of DNA with increasing
amounts of AO. When the concentration of AO becomes equal to 84 × 10-8 mol L-1 the
same peak at +0.86 V appears, which declares the result of the electrostatic binding
between the two components.

Acridine orange is bound to the electrode surface

It is already proven that DNA, RNA and other biomacromolecules can be strongly
adsorbed at the mercury and carbon paste electrodes. So, it is possible to prepare a
DNA-modified electrode by dipping the electrode into the DNA solution for a short
time and they washing it. Low-molecular mass substances do not have the tendency
to adhere to the surface of the electrodes or they are removed by washing. In some
cases strong binding of low-molecular mass compounds, such as mitomycin or dauno-
mycin, to the electrode surface was observed due to chemisorption, polymerization,
condensation, etc. of the given substance [15,24,25]. AO presents a well-developed
peak obtained with CPE which was first pretreated and immersed into the solution of
2 × 10-7 mol L-1 AO for 2 min at +0.5 V, washed and transferred into a blank back-
ground electrolyte. This was the way to prepare an acridine orange-modified elec-
trode and to use it for the study of the surface-confined AO with DNA in solution.

Interaction of the surface-confined AO with DNA in solution.

The acridine orange-modified electrode was immersed into a solution of dsDNA
at a final concentration ranging from 0 to 90 mg L-1. The incubation time was 2 min at
a potential of +0.2 V. As a result of this procedure, two peaks appeared possibly due
to the interaction of dsDNA and AO; the one at +0.85 V, which characterizes the
complex, and the other at +0.99 V. By increasing the amount of dsDNA into the
solution, the peak at +0.86 V increases and becomes well-defined. But the peak height
is not so high because of the smaller quantities used compared to the other interac-
tions.
The immobilization of AO at the electrode surface did not prevent the interaction
of AO with dsDNA and that the resulting complex appears also at a similar potential,
leading to the conclusion that AO is capable of interacting with dsDNA molecules
diffusing to the surface from the bulk solution. If AO was attached to the electrode
surface due to its polymerization or chemisorption, no interaction with DNA would
occcur. Further studies could be done on the different positioning of the DNA mol-
ecule at the electrode surface due to the modification of the electrode surface with
AO.
478 I.Ch. Gherghi, S.Th. Girousi, A.N. Voulgaropoulos and R. Tzimou-Tsitouridou

Immobilization of poly(G) at the surface electrode and interaction

with AO in solution
In order to study the selectivity of the interaction between DNA and acridine
orange a poly(G)-modified electrode was prepared. This electrode was prepared by
immersing the CPE into a 0.2 mol L-1 acetate buffer solution, of pH 5.0, which con-
tained the synthetic polyribonucleotide poly(G) at a final concentration of 0.1 g L-1
for 2 min at +0.5 V. The electrode was washed and immersed into solutions of AO
with different concentrations ranging from 0 to 75 × 10-8 mol L-1. Figure 9 shows the
dependence of the peak current of the characteristic peak of poly(G) at +0.994 V on
the concentration of AO added. It has to be mentioned that the characteristic peak of
the complex formed between AO and poly (G), started appearing at the concentration
of 4 × 10-8 mol L-1. The latter peak is less intense compared to the others coming from
the complexation of dsDNA and AO or ssDNA and AO.

1200 2
1100
1000
1
2
900
800
700
600
I, nA

500
400
300
200
100
0
-100
-10 0 10 20 30 40 50 60 70 80
-8 -1
C[AO] ( x10 mol.L )

Figure 9. 1. Dependence of peak current of oxidation of guanine residues in poly(G) immobilized on the
electrode surface on concentrations of AO. The CPE was pretreated at +1.7 V for 1 min followed
by adsorptive accumulation of poly(G) at +0.5 V for 2 min and then was immersed in AO solu-
tions of different concentration; the incubation time prior to each scan was 2 min; 2. Dependence
of peak current probably due to AO on increasing concentrations of AO into the solution. The
oxidation potential is at +0.79 V
 DNA modified carbon paste electrode 479

In this case, we cannot speak about intercalation, since poly(G) is a ribonucleo-

tide which tends to form a four-stranded structure, but we may assume that there is an
interaction between the two of them. We can conclude about the selectivity of the
binding, which is helpful in designing sequence selective sensors (DNA hybridiza-
tion sensors).

CONCLUSIONS

In this paper we have shown that the intercalation of AO to DNA immobilized at

the CPE surface can be monitored by adsorptive transfer stripping voltammetry with
differential pulse mode.
In conclusion, we have applied a dsDNA and a ssDNA-modified electrode which
can be used in the detection of AO [26]. By using the DNA-modified electrode and
monitoring the changes of the characteristic peak of guanine, the determination of
very low concentrations of AO is efficient. In addition, the differentiations of the
interaction between dsDNA or ssDNA with a potent intercalator could be used as a
criterion for the intercalation phenomenon.
The study of these changes and the different way of action with dsDNA or ssDNA
could highlight the mechanism of the interaction between different chemical com-
pounds. These results could also be useful for the development of an electrochemical
DNA hybridization sensor. New indicators are needed in order to improve the sensi-
tivity of the electrochemical methods, but they should be characterized by very low
adsorption at the electrode surface. Finally, we have shown that the adsorptive trans-
fer stripping voltammetry with the differential pulse mode can be used in order to
study the differences in the interaction of AO with dsDNA, ssDNA and poly(G) and
explore the mechanism of DNA intercalation.

Acknowlegements

The authors wish to thank the Laboratory of Biochemistry of the Aristotle University and particu-
larly the Director Prof. D. Kyriakidis and the Lecturer Dr. A. Pantazaki for the initial discussions regar-
ding the project.

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Received February 2003

Accepted August 2003

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