Simultaneous determination of guanine and adenine in DNA ...

Analytica Chimica Acta 461 (2002) 243–250

Simultaneous determination of guanine and adenine in DNA using
an electrochemically pretreated glassy carbon electrode

Huai-Sheng Wang, Huang-Xian Ju, Hong-Yuan Chen*

State Key Laboratory of Coordination Chemistry, Department of Chemistry, Institute of Analytical Science,
Nanjing University, Nanjing 210093, PR China

Received 19 July 2001; received in revised form 20 March 2002; accepted 2 April 2002

Abstract

A simple and reliable method based on adsorptive stripping at an electrochemically pretreated glassy carbon electrode
(GCE) was proposed for simultaneous or individual determination of guanine and adenine in DNA. The detection sensitivity
of guanine and adenine was improved greatly by activating the GCE electrochemically. After accumulation on pretreated GCE
at open circuit for 5 min or at the potential of +0.3 V for 120 s, guanine and adenine produced well-defined oxidation peaks at
about +0.8 and +1.1 V, respectively in pH 5 phosphate buffer. The detection limit for individual measurement of guanine and
adenine was 4.5 ng ml−1 (3 × 10−8 mol l−1) and 4 ng ml−1 (3 × 10−8 mol l−1), respectively. Acid-denatured DNA showed
two oxidation peaks corresponding to guanine and adenine residues in the same buffer. The proposed method can be used to
estimate the guanine and adenine contents in DNA with good selectivity in a linear range of 0.25–5 ␮g ml−1. © 2002 Elsevier
Science B.V. All rights reserved.

Keywords: Guanine; Adenine; DNA; Adsorptive stripping voltammetry; Glassy carbon electrode; Electrochemical pretreatment

1. Introduction trochemical detection [7–9], have been developed for
the detection and quantification of purine bases in nu-
Guanine and adenine are important components cleic acids. In addition, voltammetric techniques are
found in deoxyribonucleic acid. Determining indi- also suitable for the analysis of these purines in nu-
vidual concentrations of guanine and adenine or cleic acids [10], and some methods for the determina-
their ratio in DNA is important to the measurement tion of guanine and adenine based on their oxidation
of nucleic acid concentration itself. Indeed, nucleic at pyrolytic graphite, glassy carbon, and carbon paste
acid components in physiological fluids, tissues and electrodes have been developed [11–16]. Compared
cells are related to the catabolism of nucleic acids, to the spectroscopic methods, electrochemical meth-
enzymatic degradation of tissues, dietary habits, and ods reported for guanine and adenine measurement
various salvage pathways. Therefore, detection of ele- suffer from problems of irreversible adsorption of
vated levels of these substances could be indicative of these purine bases on the electrode surface [12–14].
certain disease [1]. Many methods, such as the spec- Oliveira Brett and Matysik used sonovoltammetry
troscopic methods coupled with chromatography or [16] to control the extent of the adsorption of the
electrophoresis [1–6] and electrophoresis with elec- relevant species and obtained a detection limit of
2 × 10−7 M for guanine. A differential pulse adsorp-
∗ Corresponding author. Fax: +86-253317761. tive stripping voltammetry has also been developed in
E-mail address: [email protected] (H.-Y. Chen). our laboratory for the detection of guanine by forming

0003-2670/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 0 0 3 - 2 6 7 0 ( 0 2 ) 0 0 2 9 7 - 0

244 H.-S. Wang et al. / Analytica Chimica Acta 461 (2002) 243–250

a complex with Hg [17]. Recently, Zen et al. re- 2. Experimental
ported the simultaneous determination of guanine and
adenine using a chemically modified electrode [18]. 2.1. Chemicals and apparatus

Carbon electrodes have been widely used to con- Adenine and guanine were purchased from Sigma.
struct DNA biosensors or DNA-modified electrodes Calf thymus DNA was obtained from Sano-American
[19–24], but have been little used for the direct de- Biology Company and was used without further
termination of nucleic acid [25–27] because of poor purification. All other chemicals were of analytical
voltammetric peaks for guanine and adenine residues reagent grade. The double distilled water was used
in DNA and RNA chains [28,29]. Recently, Wang for all experiments.
et al. [30,31] reported well-developed oxidation
peaks for DNA and RNA at electrochemically pre- Cyclic and linear sweep voltammetric studies
treated carbon paste electrodes using constant-current were carried out using 270 Electrochemistry System
chronopotentiometry and sophisticated baseline cor- (EG&G, USA). The three-electrode system used in
rection techniques. The stripping analysis of nucleic this study contained a glassy carbon working elec-
acids was reported for electrodes of various carbon trode, a platinum wire counter electrode and a SCE
materials, with detection limits of 20, 30, 40 and reference electrode.
50 ng ml−1 for oligo(dG)20 obtained at carbon paste,
pyrolytic graphite, highly oriented pyrolytic graphite, 2.2. Pretreatment of the glassy carbon electrode
and carbon strip electrodes, respectively. However,
no response was observed at a glassy carbon elec- GCE (0.07 cm2; BAS, USA) was polished to mir-
trode (GCE) under the experimental condition em- ror finish using the BAS-polishing kit with 0.3 and
ployed [32]. With differential pulse voltammetry, a 0.05 ␮m alumina paste. After rinsed with distilled wa-
large difference in the oxidation signals of native and ter thoroughly, the electrode was applied a potential
acid-denatured DNA was obtained at GCE [33]. This of +1.80 V under stirring in 0.1 M pH 5 PB for 300 s,
difference resulted from the deep DNA degradation and then the electrode was scanned between +0.3 and
involving DNA depurination [34]. Electrochemical +1.25 V until a steady-state current–voltage curve
pretreatment of GCEs has been widely used to im- was obtained. After this treatment, a thin blue film
prove electrode response [35–41]. In our previous can be observed on the activated electrode surface.
work [35], a difference between native and thermally No significant difference of response of guanine at
denatured single-stranded DNA was obtained at the the pretreated GCEs was observed between different
pretreated GCE. This electrode showed a linear re- GCEs that were from BAS and Jiangsu Electroana-
sponse to the thermally denatured ssDNA in the lytical Instrument Factory (China). The response of
concentration range of 6–60 ␮g ml−1. guanine at the pretreated GCE has no obvious change
after the electrode was stored in pH 5 phosphate
Guanine and adenine electro-oxidation is useful for buffer solution (PBS) for 2 days.
the detection of DNA and the design of DNA biosen-
sors based on the oxidation of guanine and adenine 2.3. Preparation of DNA samples
[42–45]. This work describes the electrochemical
oxidation of these species at an electrochemically A gentle treatment of DNA with 1 mM HCl leads to
pretreated GCE and develops a simple, reliable, and the selective removal of its purine bases by cleavage
inexpensive method for the individual or simultane- of purine glycoside bounds [46]. The calf thymus ds-
ous determination of guanine and adenine in DNA DNA was hydrolyzed as follows for quantification of
at low levels. The results indicated that the adsorp- guanine and adenine. Three milligrams of dsDNA was
tion of guanine and adenine was greatly enhanced at digested using 1 ml 1 M HCl in a sealed 10 ml glassy
the electrochemically pretreated GCE. Thus, guanine tube. After heating in a boiling water bath for 80 min,
and adenine could be accumulated on the pretreated the solution was adjusted with 1 ml 1 M NaOH. Then
electrode surface at a suitable potential or at open the solution was diluted to 10 ml using 0.1 M pH 5
circuit to produce a 10-fold increase in the oxidation PBS. For comparison, the dsDNA was digested with
signals.

H.-S. Wang et al. / Analytica Chimica Acta 461 (2002) 243–250 245

perchloric acid according to [12]. Three milligrams (dotted line) and pretreated GCE (solid line) in 0.1 M
of dsDNA was dissolved in 0.5 ml concentrated per- pH 5 PBS. At the pretreated GCE, the guanine and ade-
chloric acid and stirred for 10 min, then 0.5 ml 9 M nine give two well-defined oxidation peaks in the first
NaOH was added to neutralize the solution followed anodic sweep at about +0.8 V (peak G) and +1.1 V
by adding 5 ml of 0.2 M pH 5 PBS. This solution was (peak A), while the response at the unpretreated GCE
diluted with water to the final volume of 10 ml. is very poor. The background current of GCE showed
almost no increase after the pretreatment. The peak
2.4. Voltammetric procedure potentials of guanine and adenine at the pretreated
GCE were slightly more negative than those obtained
The electrochemical experiments were performed at unpretreated GCE. No reduction peak is observed
in 0.1 M pH 5 PBS with different concentrations of in cathodic sweep, thus, the electrochemical oxidation
guanine and adenine. The adsorptive accumulation of of guanine and adenine on pretreated GCE are entirely
guanine and adenine at the working electrode was done irreversible process.
in a stirred solution at +0.3 V for 120 s or at open
circuit for 5 min. After a 5-s quiet period, the anodic 3.2. Adsorption of guanine and adenine
stripping voltammograms were recorded from +0.3
to +1.25 V at 100 mV s−1. The electrode can be used When immersing the pretreated electrode into a
for next measurement after continuous sweep for three quiet phosphate buffer (pH 5) containing guanine and
cycles at the same potential range. adenine for 5 min, the cyclic voltammogram shows
very low responses. After a 5 min stirring at open cir-
3. Results and discussion cuit, however, the responses improved greatly. After
the pretreated electrode is immersed in the stirring
3.1. Cyclic voltammograms of guanine and adenine solution containing guanine and adenine for 5 min
at glassy carbon electrode and transferred it to pH 5 PBS, the peaks of guanine
and adenine can be observed. This suggests that gua-
Fig. 1 shows the cyclic voltammograms of 20 ␮M nine and adenine can be adsorbed on the pretreated
guanine and 20 ␮M adenine at the unpretreated GCE electrode surface. It can be seen from Fig. 1 that the
peaks of guanine and adenine almost disappeared at
the second cycle. This phenomenon may be partly
attributed to the consumption of adsorbed guanine
and adenine and the adsorption of electrochemical
oxidation products at the pretreated GCE surface.

3.3. Effect of pH and scan rate (ν)

Fig. 1. Successive cyclic voltammetric sweeps in 0.1 M pH 5 PBS Both the peak current and potentials were depended
containing 20 ␮M guanine and 20 ␮M adenine at unpretreated
on pH values of solution. The pH dependence of ox-
GCE (dotted line) and pretreated GCE (solid line). Accumulated
at +0.3 V for 120 s and the scan rate is 100 mV s−1. idation peak potentials of guanine and adenine obeys
the equations, Ep = 1.068 − 0.049pH (peak G, r =
0.9993) and Ep = 1.429 − 0.060pH (peak A, r =
0.9992), respectively. The oxidation of guanine and
adenine themselves follows a two-step mechanism in-
volving the total loss of 4e− and the first 2e− oxida-

tion is rate-determining step [47]. The slopes of 49
and 60 mV pH−1 show that two protons take part in

the rate-determining step.

The peak currents and potentials of cyclic voltam-

mograms of the pretreated electrode in guanine

246 H.-S. Wang et al. / Analytica Chimica Acta 461 (2002) 243–250

and adenine solution (pH 5) vary with change 3.4. Determination of guanine and adenine
of scan rate. In the range of 25–150 mV s−1, the
relations obey the following questions, log ip = 3.4.1. Individual determination of guanine
−1.226 + 0.719 log ν (peak G, r = 0.9994) and and adenine
log ip = −1.423 + 0.737 log ν (peak A, r = 0.9998)
(ν in mV s−1 and ip in mA). This indicates that the At the experimental conditions, the calibration
electrode process was controlled simultaneously by curve of guanine showed two linear regions with
different slopes. The initial linear portion increases
diffusion and adsorption. With increasing scan rate, from 0.08 to 1 ␮M with a regression equation of ip =
1.95 × 106C (r = 0.9995, C in M). The second linear
both anodic peak potentials shifted to more positive segment up to 2.72 ␮M with a regression equation of

values.

Fig. 2. Linear sweep stripping voltammograms of various concentrations of adenine from 0.16 to 6.10 ␮M in 1.92 ␮M guanine solution.
Inset: plot of peak currents ((᭹) for adenine, (᭿) for guanine) vs. adenine concentrations. Other conditions were the same as in Fig. 1.

H.-S. Wang et al. / Analytica Chimica Acta 461 (2002) 243–250 247

ip = 0.69 + (1.18 × 106C) (r = 0.9980). They can be of 1 ␮M adenine. The detection limit of 3 × 10−8 M
used to determine the guanine at different concentra- (4 ng ml−1) and the relative S.D. of 3.5 and 4.5% for
tions. The detection limit of 3 × 10−8 M (4.5 ng ml−1) 1.44 and 0.64 ␮M adenine were obtained (n = 6).

was obtained with the accumulation time 240 s. The 3.4.2. Simultaneous determination of guanine
and adenine
relative S.D. of 3.2 and 4.4% for 1.44 and 0.64 ␮M
guanine (n = 6) showed good reproducibility. As to Fig. 2 shows the linear sweep stripping voltam-
adenine, in the range of 0.08–2.72 ␮M, the same trend mograms when 1.92 ␮M guanine and adenine with
the concentrations from 0.16 to 6.1 ␮M coexisted in
of calibration curve was obtained with the regression solution. Although there is a competitive adsorption
equations of ip = 0.22 + (1.96 × 106C) (r = 0.9991)
and ip = 1.16+(1.02×106C) (r = 0.9975) at the turn

Fig. 3. Linear sweep stripping voltammograms of various concentration of guanine from 0.16 to 5.44 ␮M in 1.92 ␮M adenine solution.
Inset: plot of peak currents ((᭹) for guanine, (᭿) for adenine) vs. guanine concentrations. Other conditions were the same as in Fig. 1.

248 H.-S. Wang et al. / Analytica Chimica Acta 461 (2002) 243–250

at GCE, the linear relation can be observed at low from a mixture at the concentration range studied by
concentrations of adenine up to 1 ␮M while the peak the proposed method.
current of guanine holds at stable value. The increase
of adenine concentration leads to the decrease of peak 3.4.3. Analytical application
current of guanine. When 1.92 ␮M adenine coexisted The acid-denatured DNA gives two well-defined
with various concentration of guanine, the same trend
can be observed (Fig. 3). These results indicate that peaks at the pretreated GCE due to the oxidation of
the competitive adsorption equilibrium can be attained guanine and adenine residues. Fig. 4 shows the typ-
at the suitable concentrations of guanine and adenine. ical LSV responses of guanine and adenine residues
Therefore, the guanine and adenine could be estimated with increasing DNA concentration from 0.50 to
25 ␮g ml−1. The relationship between the peak

Fig. 4. Linear sweep stripping voltammograms for the simultaneous determination of guanine and adenine in acid-denatured DNA with
increasing concentration from 0.50 to 25 ␮g ml−1. Inset: plot of the peak currents vs. acid-denatured DNA concentrations. Other conditions

were the same as in Fig. 1.

H.-S. Wang et al. / Analytica Chimica Acta 461 (2002) 243–250 249

Table 1 Acknowledgements
Content of guanine and adenine in DNA simultaneously determined
with a pretreated GCE (n = 4) The financial support from the National Sci-
ence Foundation of China (nos. 29835110 and
Calf thymus DNA 29975013), the Science Foundation of Jiangsu (No.
BK99030), the Foundation of the Education Ministry
Guanine (mol%) Treated by Treated of China (Nos. 200028403 and 2000143) and the
Adenine (mol%) HClO4 by HCl State Key Laboratory of Electroanalytical Chemistry,
Molar ratio ((G + C)/(A + T)) Changchun Institute of Applied Chemistry are greatly
22.52 21.34 acknowledged.
27.48 28.66

0.82 0.74

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