ELSEVIER
Journal of Electroanalytical Chemistry 429 (1997) 95-99
Voltammetric and sonovoltammetric studies on the oxidation of thymine
and cytosine at a glassy carbon electrode
Ana Maria Oliveira Brett *, Frank-Michael Matysik
Departamento de Qufmica, Faculdade de Ci~ncias e Tecnologia, Universidade de Coimbra, 3000 Coimbra, Portugal
Received 13 August 1996; revised 21 October 1996
Abstract
The voltammetric behaviour of the pyrimidine bases thymine and cytosine is studied using a glassy carbon electrode. In contrast to
previous reports that assume electroinactivity at carbon electrodes, it is demonstrated that both compounds undergo well-defined oxidation
at a glassy carbon electrode. The experimental conditions that influence the electrode reaction, in particular the pH of the electrolyte
solution, are varied systematically and optimized for voltammetric determinations. The application of ultrasound in combination with
differential pulse voltammetry results in a reliable analytical procedure for thymine and cytosine measurements avoiding electrode fouling
and maintaining the electrode characteristics. The effect of ultrasound is mainly to enhance transport of electroactive species and to clean
the electrode in situ. Besides studying the sonovoltammetric behaviour of cytosine and thymine separately, simultaneous determinations
are also performed and extended to the analysis of adenine, guanine, thymine and cytosine in the same solution. © 1997 Elsevier Science
S.A.
Keywords: Cytosine; Thymine; Sonoelectrochemistry; Ultrasound; Voltammetry
1. Introduction
Pyrimidine and purine derivatives play an essential role
in various biological processes. In particular, the nucleotides of thymine and cytosine together with those of
adenine and guanine represent the monomer units of nucleic acids. The genetic information of deoxyribonucleic
acid (DNA) is determined by the sequence of the purine
and pyrimidine bases, whereas ribose and phosphate groups
of the nucleotide units have a structural role.
Investigations of the redox behaviour of biologically
occurring compounds by means of electrochemical techniques have the potential for providing valuable insights
into biological redox reactions of such biomolecules. In the
case of the pyrimidine bases thymine and cytosine several
studies have been undertaken concerning their redox behaviour at mercury electrodes. Cytosine was found to be
reducible at the mercury electrode [1-4], whereas thymine
was reported to show no polarographic reduction wave
[1,2]. In addition, both bases form sparingly soluble compounds with the mercury electrode which allows their
* Corresponding author.
0022-0728/97/$17.00 © 1997 Elsevier Science S.A. All rights reserved.
PII S0022-0728(96)05018-8
determination by cathodic stripping voltammetry [5]. However, according to previous literature reports cytosine and
thymine have been assumed to exhibit no electrochemical
activity at graphite electrodes [3,6,7] or carbon based
electrodes in general [8,9].
During the course of this work we found that both
pyrimidine bases undergo oxidation at a glassy carbon
electrode. The present paper deals with the optimization of
the conditions that influence the voltammetric response,
e.g. the pH of the electrolyte solution and the conditioning
of the electrode surface. Ultrasonic pretreatment of the
glassy carbon electrode was utilized in order to permit
reliable voltammetric determinations of thymine and cytosine. A recently developed small-volume sonovoltammetric cell [10] in which the glassy carbon electrode is exposed to high intensity ultrasound was found to be suitable
for these studies. The main effects of ultrasound that can
be exploited for electrochemical purposes are the enormous enhancement of mass transport [11-13] and electrode surface modification or cleaning which helps to
avoid progressive electrode fouling [14,15]. The latter aspect was found to be particularly useful in the present
study in order to enhance the reliability of cytosine and
thymine determinations.
96
A.M. Oliveira Brett, F.-M. Ma~.'sik/ Journai of Electroanalytical Chemistry 429 (1997) 95-99
As a result of separate studies on the voltammetfic
behaviour of cytosine and thymine, optimized conditions
were selected for the determination of both compounds in
the same solution and it will be demonstrated that even all
four DNA bases, i.e. adenine, guanine, thymine and cytosine, can be determined simultaneously by ultrasound-assisted differential pulse voltammetry.
2. Experimental
The cell configuration used for the sonovoltammetric
experiments has been described in detail in a previous
paper [10]. The thermostatted glass cell (25°C) contained
20 ml electrolyte solution. A glassy carbon electrode (GCE)
of 6 mm diameter was positioned at the bottom of the cell
so as to face the tip of the sonic horn at precisely measured
and calibrated distances. A platinum coil was used as a
counter electrode and a laboratory-made silver[silver chloridel3 M KCi electrode served as the reference electrode.
The horn was connected to a tapered microtip (d = 3 mm)
fabricated from high grade titanium alloy. The ultrasonic
processor was a Model VCS01 (Sonics and Materials Inc.,
USA) capable of delivering up to 500W at 20kHz frequency. The ultrasonic processor L, d:'signed to deliver a
constant amplitude that can be selected via the amplitude
control setting, ranging from 0 to 100; however, in conjunction with the microtip the amplitude control setting
must not be higher than 40. The actual power intensity
entering the system was calibrated calotimetrically according to the procedure of Mason et al. [16]. For relevant
amplitude control settings of 10, 15 and 20 the corresponding power intensities were 16 + 3, 30 + 3 and 72 +
5 W cm-2 respectively. The sonovoltammetric cell and the
sonic horn were housed in a sound-proofed cage in order
to protect the operator from high-intensity acoustic noise.
All voltammetric experiments were done using an Autolab PGSTATI0 potentiostat (Eco Chemie, Utrecht, Netherlands) equipped with an ECD low current module. The
current signal was filtered through a third order Sallen-Key
filter with a time constant of 0.1 s in order to remove
high-frequency a.c. components. The pH values of electrolyte solutions were measured using a Crison Model
micropH 2001 pH-meter and an Ingold combination glass
electrode.
The GCE (a gift from Professor G. Jenkins, A&M
University, Normal, Alabama, USA) was prepared for
measurement by polishing using plastic foils (Hirschmann,
Germany) with adherent alumina of decreasing particle
size ranging from 9 to 0.3 Ixm, followed by thorough
rinsing with Milli-Q water. Prior to recording voltammograms of electroactive species, several cyclic voltammograms were performed within the same potential limits in
the background solution until a stable voltammetric response was obtained.
Adenine, guanine, thymine, cytosine, guanosine, adeno-
sine, thymidine, cytidine and the mono-, di-, and triphosphates of thymidine and cyddine were obtained from
Sigma Chemical Co. and used as received. All solutions
were made using high-purity water from a Millipore MilliQ system (resistivity > 18 M II cm) and analytical-reagent
grade chemicals.
The following supporting electrolytes were used: 0.1 M
perchloric acid, acetate buffer solutions containing 0.1 M
sodium acetate/acetic acid covering the pH range between
4.0 and 5.5, 0.1 M phosphate buffer solutions (pH 6.5 to
7.5), 0.1 M borate/sodium hydroxide solutions for the pH
range between 9.2 and 12.3, and a 0.1 M sodium hydroxide
solution. The purine and pyrimidine derivatives were dissolved directly in the buffer solutions except in the case of
guanine. Stock solutions of I mM guanine were made
either in 0.1 M NaOH or in 0.1 M HCIO4. Working solutions of guanine were prepared by adding small volumes
of stock solution to the corresponding buffer solution. The
solutions were then sonicated in order to ensure homogenization.
3. Results and discussion
Initial studies of the voltammetric behaviour of cytosine
and thymine were performed in acetate buffer (pH 4.50).
Cyclic voltammograms were recorded in the absence and
presence of cytosine and thymine respectively. In the case
of cytosine an increase in current occurred at the positive
limit of the accessible potential range (ca. 1.45 V), whereas
thymine already showed a well-defined oxidation wave
with a half-wave potential of 1.27 V. The pH dependence
of the oxidation of both pyrimidine bases was studied
systematically in the pH range between 1 and 13. In 0.1 M
HCIO4 only thymine gave an oxidation signal; cytosine
was not oxidizable at the glassy carbon electrode within
the accessible potential region. Fig. 1 illustrates the dependence of differential pulse peak potentials of cytosine and
1.5-"
1.41.3-
>
1.2
1.1
1.0
O.9
0.7
~
~
1'0
~'2
~
pH
Fig. 1. Dependence of differential pulse (DP) peak potential of thymine
(Q) and cytosine (ll) on pH of the supporting electrolyte solution.
Thymine and cytosine concentrations 5× 10-4 M. Scan rate 5mVs -l,
amplitude 50mV.
A.M. Oliveira Brett, F.-M. Matysik / Journal of Electroanalytical Chemistry 429 (1997) 95-99
A
I
,
I
0.2
,
I
0.4
0.6
t
i
.
I
0.8
.
I
1.0
,
I
1.2
"':.4
E/V
',,/(1)I
, f " / , ~'2)1
B
/
.,llO)l
lO0
(b)
,
0.2
0.4
..a..---
0.6
,
0.8
.
.
1.0
1.2
1.4
EIV
Fig. 2. Cyclic voltammograms at a glassy carbon electrode in borate
buffer: (A) 2.6 mM thymine (pH 10.0) and (B) 5 mM cytosine (pH 10.79).
Scans (1) to (4) are successive recordings, (b) background response. Scan
rate 50mVs -t.
thymine on pH. For thymine the slope of the Ep-pH plot
is - 6 0 mV per pH unit over the whole pH range studied,
which suggests that the number of protons and electrons
involved in the oxidation mechanism is equal. In the case
of cytosine we find a change in slope of the Ep-pH plot at
about pH 10. For pH values lower than 10 the slope is
- 6 0 m V per pH unit and in the more alkaline region
aEp/0pH is - 8 5 mV per pH unit. This indicates that the
ratio of the number of protons and transferred electrons
shifts at pH 10 from 1 to 3/2. This could be explained if,
for example, a product of oxidation undergoes deprotona-
~
1
oo w~
(1)
97
tion at pH > 10 while the prote,l}~ic state of the educt
remains as it was at lower pH. Delia et al. [ 17] reported the
formation of cytosine 3-N-oxide by homogeneous reaction
between cytosine and m-chloroperbenzoic acid. They determined pK values of 4.82 and 10.3 for cytosine 3-Noxide, as comp,'u'ed with 4.60 and 12.16 for cytosine.
Speculating that cytosine 3-N-oxide is the oxidation product formed voltammetrically, this could explain the change
in slope of the Ep-pH function for cytosine as discussed
above, because cytosine 3-N-oxide becomes deprotonated
at pH higher than 10.3. However, there is no proof that the
electrochemical oxidation follows the same mechanism.
More work has to be done concerning the analytical
identification of the products of electro-oxidation of
thymine and cytosine respectively.
From the results obtained by studying the pH dependence of the oxidation potentials of cytosine and thymine,
optimum pH values were selected for further characterization by cyclic voltammetry. Fig. 2 shows the cyclic
voltammetric responses of both compounds. The oxidation
signals are well resolved from the background response. In
contrast, the nucleosides and nucleotides of thymine and
adenine were found not to be oxidizable under the same
conditions. The cyclic voltammograms shown in Fig. 2
exhibit an obvious signal decrease during successive
recordings. This behaviour is similar to that of the corresponding purine compounds [15] and results from the
adsorption of oxidation products that block the electrode
surface.
The situation can be improved considerably by applying
ultrasound while recording the cyclic voltammogram. This
is illustrated in Fig. 3 where successive cyclic sonovoltammograms of thymine are presented that show no tendency
to signal decrease. However, the limiting current region of
the sonovoltammograms is not very extended and consequently not well suited for quantitative evaiuation. Therefore, the analytical procedure for quantitative determinations of thymine and cytosine is based on ultrasound-assisted differential pulse voltammetry (DPV) where ultrasound is applied (1 to 2 min) but is switched off in the
(2)
(3)
(1)
, (2)
, (3)
, (4)
2o
, (5)
,
/
y/)'/y
.a...--
'o'. 4
0.8
........
1.0
1.2
1.4
EIV
Fig. 3. Cyclic sonovoltammograms of 1 × 10-4M thymine in borate
buffer (pH 10.0) at a glassy carbon electrode. Scans (1) to (3) represent
successive sonovoltammograms. Ultrasound conditions: 5 mm horn tipelectrode separation, power intensity 30Wcm -2. Scan rate 50mVs-
I
I
I
0.8
1.0
1,2
E/V
Fig. 4. Repetitive ultrasound-assisted differential pulse voltammograms of
5x IO-4M cytosine in borate buffer (pH 10.79). Ultrasound (power
intensity 30Wcm-2 horn tip-electrode separation 6 mm) is applied until
0.8 V. The potential window is given for voltammogram (3). DP conditions as in Fig. 1.
98
A.M. Oliveira Brett. F.-M. Ma~. sik / Joun~al of Electroanalytical Chemistry 429 (1997) 95-99
~
o~
02
,
0,
,
0'6
,
0'8
,
I
,
i2
I
,
I
/////~
1
I
I
I
I
0.4 0.6 0.8 1.0 1.2 1.4 16
E/V
EIV
Fig. 5. Ultrasound-assisteddifferential pulse voltammetric determinations
of thymine and cytosine in borate buffer (pH 10.02). Ultrasonic pretreatment (power intensity 30Wcm -2, horn tip-electrode separation 5 mm)
until 0.75V. Concentrations: (!) background response, (2) 5x lO-4M
thymine, (3) 5 x l O - 4 M thymine and 2.5x lO-4M cytosine, (4) 5x
10-4 M thymine and 5 x 10-4 M cytosine. DP conditions as in Fig. !.
relevant potential region of the differential pulse voltammetric recording in order to avoid negative effects on the
precision of the signal due to ultrasonically induced mass
transport fluctuations. By means of this analytical strategy
excellent reproducibility is obtained, as illustrated for
repetitive determinations of cytosiPe shown in Fig. 4. No
electrode fouling effects occur over measuring periods of
several hours. The differential pulse voltammetric response
of thymine is similar and an even more symmetrical peak
is obtained because the signal appears at about 200mV
~ess positive potentials. Results of calibration measurements are shown in Table 1, which demonstrate the utility
of ultrasound-assisted DPV for quantitative determinations
of thymine and cytosine. The limit of detection (defined as
the concentration that leads to a signal which is three times
the standard deviation of the baseline noise) is 1 × 10-5 M
for both compounds.
The simultaneous determination of thymine and cytosine was performed at pH 10, which is the optimum with
respect to peak separation. Fig. 5 shows ultrasound-asbisted DPV recordings for a constant concentration of
thymine in the absence and at two different concentrations
of cytosine. The simultaneous quantitative determination
of both compounds is possible, but the effect of cytosine
on the signal shape of thymine has to be taken into
consideration.
Fig. 6. Differential pulse voltammograms of 6x 10-SM adenine and
5 × 10-5 M adenosine. Voltammograms(I), (2), (3) and (6) are recorded
in combination with ultrasonic pretreatment, (4) and (5) without ultrasonic pretreatment. Arrows indic;,te the potential where the ultrasound is
switched off; ultrasound conditions: 2 mm horn tip-electrode separation,
power intensity 30Wcm-2, DP conditions as in Fig. !. Supporting
electrolyte acetate buffer (0. ! M, pH 4.50).
This procedure was tested for the determination of all
four DNA bases, i.e. adenine, guanine, thymine and cytosine in the same solution by performing just one differential pulse voltammetric run. The sonovoltammetric behaviour of guanine has been reported in detail in a previous paper [15], which demonstrates the sonovoltammetric
determination of guanine. Cyclic sonovoltammograms of
adenine (not shown) indicate that there are more serious
problems of electrode blocking effects than in the case of
guanine, at least for adenine concentrations higher than
l0 -5 M. However, by means of ultrasound-assisted DPV
which allows the selection of higher power intensities
a n d / o r closer horn tip-electrode separations, reliable adenine determinations are possible up to adenine concentrations of 10 -4 M. For example, Fig. 6 illustrates repetitive
determinations of adenine in the presence of the corresponding nucleoside adenosine by DPV. It is obvious from
Fig. 6 that those DPV recordings after ultrasonic pretreatment lead to reproducible signals for adenine (Ep = 1.09 V)
and adenosine (Ep = 1.30 V). In contrast, as demonstrated
by recordings (4) and (5) of Fig. 6 without ultrasonic
pretreatment, the DP signals tend to decrease and even an
additional peak (Ep = 0.86V) resulting from some adsorbed product occurs in scan (5).
Table !
Results of linear regression of calibration data for cytosine and thymine determination by means of ultrasound-assisted DPV. Concentration range
5 x 10-5 to 5 x 10-4 M for both compounds. Experimental conditions as in Fig. 5
Analyte
thymine
cytosine
Buffer
borate buffer (0.1 M, pH 10.02)
borate buffer (0.1 M, pH 10.79)
'~;lope/gA gM-i
0.031
0.051
Intercept/izA
-0.40
0.11
Regression coefficient
0.9986 (n = 10)
0.9998 (n = 10)
m
A.M. Oliveira Bretl, F,-M. Matysik / Journal of Electroanalytical Chemistry 429 (1997) 95-99
T
/
. I
tl
~
'-~..,'--~
,_,
/;7,"
(b>
(a) -----""~
0.0
0.2
. . . .
0.4
0.6
0.8
1.0
1.2
1.4
99
show no electrochemical activity at carbon electrodes
[3,6-9].
The problem of progressive electrode fouling encountered when performing repetitive determinations of thymine
and cytosine has been solved by applying ultrasound-assisted DPV. On the basis of this approach cytosine and
thymine can be reliably determined either separately or in
a mixture.
Finally, it has been shown that all four DNA bases,
adenine, guanine, thymine and cytosine, can be measured
simultaneously by ultrasound-assisted DPV. This is obviously a clear advantage over measurements based on the
dropping mercury electrode which does not offer such
versatility in the context of the analysis of DNA bases.
EIV
Fig. 7. Differential pulse voltammetric determination of purine and
pyrimidine bases guanine (2× l0 -5 M), adenine (3 x 10-5 M), thymine
(3 x l0 -4 M) and cytosine (3 × l0 -4 M) in borate buffer (pH 10.02), (a)
with ultrasonic pretreatment (power intensity 72 Wcm-2 horn tip-electrode separation 5ram), (b) successive scan without ultrasonic pretreatment. DP conditions as in Fig. 1.
Finally, a mixture of adenine, guanine, thymine and
cytosine was studied by DPV under conditions that give
the best peak separation for thymine and cytosine. Fig. 7
shows well separated signals for each compound and
demonstrates the excellent multi-component capability of
the proposed method. The difference between the DPV
recordings 7(a) and 7(b) is that the latter was measured
without ultrasonic pretreatment. It can be seen from Fig. 7
that the adenine and guanine peaks in 7(b) are somewhat
larger than in 7(a) and shifted slightly towards more
negative potentials. This is due to contributions to the
signal from adsorbed adenine and guanine [15]. However,
the adsorption processes are difficult to control and have
negative effects on the reproducibility, which is much
better when the voltammetric measurement is combined
with ultrasonic pretreatment.
4. Conclusions
It has been demonstrated that the pyrimidine bases
thymine and cytosine undergo oxidation at glassy carbon
electrodes, whereas previously it was assumed that they
Acknowledgements
We thank the European Community for financial support (Contract No. CHRX CT94 0475) under the Human
Capital and Mobility Scheme.
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