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www.rsc.org/analyst
Electrochemical analysis of nucleic acids at boron-doped
diamond electrodes
Csar Prado,
a
Gerd-Uwe Flechsig,
b
Peter Grndler,
b
John S. Foord,
a
Frank Marken
c
and Richard G. Compton*
a
a
Physical and Theoretical Chemistry Laboratory, University of Oxford, South Parks Road,
Oxford, UK OX1 3QZ. E-mail: compton@ermine.ox.ac.uk
b
Universitt Rostock, FB Chemie, Technische und Umweltchemie, Abt. fr Analytische,
Albert Einstein Str. 3a, D-18051 Rostock, Germany
c
Department of Chemistry, Loughborough University, Loughborough, UK LE11 3TU
Received 18th December 2001, Accepted 14th January 2002
First published as an Advance Article on the web 29th January 2002
Highly conductive boron-doped diamond (BDD) electrodes
are well suited for performing electrochemical measure-
ments of nucleic acids in aqueous solution under diffusion-
only control. The advantageous properties of this electrodic
material in this context include reproducibility and the small
background currents observed at very positive potentials,
along with its robustness under extreme conditions so
offering promising capabilities in future applications involv-
ing thermal heating or ultrasonic treatment. tRNA, single
and double stranded DNA and 2A-deoxyguanosine 5A-
monophosphate (dGMP) have been studied and well defined
peaks were observed in all cases, directly assignable to the
electro-oxidation of deoxyguanosine monophosphate.
Introduction
Nucleic acids are electroactive species that can be reduced and/
or oxidized at various electrodes. Their electrochemical activity
was discovered in the 1960s
1,2
but it has only been very
recently that the sensitivity of electrochemical analysis in this
context was increased by several orders of magnitude through
application of adsorptive stripping voltammetry
37
and chron-
opotentiometric adsorptive stripping voltammetry (CPSA)
812
using mercury electrodes. The latter shows very high sensitivity
towards the determination of nucleic acids.
13
Solid electrodes have not been used until recently due to the
large background current contributions observed at these
surfaces at the necessary potentials. Such electrodes are
attractive since they obviate the need for mercury and, in some
cases, that for deoxygenation. Accordingly chronopotentio-
metric adsorptive detection on carbon paste and pyrolytic
graphite electrodes
8,9,1418
has been successfully applied to the
study of nucleic acids with various results. Recently the use of
heated carbon paste electrodes
19,20
has shown that similar
detection limits to those found on mercury can be obtained
although classical amperometric experiments were less than
optimal, mainly due to the large background currents found at
potentials corresponding to the oxidation.
The use of boron-doped diamond electrodes
21,22
is very
attractive due to advantageous properties including high
reproducibility and stability, along with robustness under
extreme conditions where conventional electrode materials may
undergo severe erosion.
2224
BDD electrodes have also proved
very useful because they show an extremely wide potential
window in aqueous solutions without oxidation of the electrode
itself.
25
This allows electrochemical detection with only tiny
background currents of a number of substances that oxidise at
very positive potentials, where other electrodic materials are not
suitable.
It is the aim of this investigation therefore to address the
possibility of performing classical electrochemical analysis of
different nucleic acids in aqueous solutions by the use of BDD
electrodes.
Experimental
Reagents
Double stranded calf thymus DNA (dsDNA, Catalog No.
D4522), single stranded calf thymus DNA (ssDNA, Catalog
No. D8899), transfer RNA (tRNA, Catalog No. R8759) and
acetate buffer (3M pH 5.0, certified free of RNase and DNase,
Catalog No. S7899) were supplied by Sigma. 2A-deoxyguano-
sine 5A-monophosphate disodium salt (dGMP, Catalog No.
85,222-8), Tris(hydroxymethyl)-aminomethane (Tris, Catalog
No. 15,456-3) and ethylendiamine acetic acid (EDTA, Catalog
No. 10,631-3) were obtained from Aldrich. Stock solutions 1 g
L
21
of ssDNA and dsDNA were prepared in buffer (10 mM Tris
HCl, 1 mM EDTA, pH 8.0). dGMP 0.05 M and tRNA 1 g L
21
were prepared in water. Solutions were prepared using UHQ
grade water, of resistivity not less than 18 MW cm (Elgastat,
High Wycombe, UK). An electrode cleaning solution of
saturated sodium hydroxide in ethanol was used as described
below.
Electrodes
A polished BDD film (5 3 5 3 0.535 mm
3
, film resistivity 0.1
W) grown by chemical vapour deposition (CVD), supplied by
DeBeers Industrial Diamond Division (Ascot, UK), was used
without any pretreatment. This was housed in a Teflon
mounting sealed using epoxy resin Mecaprex MA2 by PRESI
(Brie, France) with an electrical connection to the rear side
made via a brass rod attached, using Silver Loaded Epoxy
Adhesive supplied by RS Components (Corby, UK). The rear of
the electrode assembly was insulated using a sealant wax and
the whole unit was introduced in a conventional three electrode
electrochemical cell.
Voltammetric procedures
Cyclic voltammograms of the supporting electrolyte alone were
explored in 20 mL volumes of 0.2 M acetate buffer solution (pH
= 5.0), as it has been found to offer the more favourable signal
to background characteristics.
15
No degassing was used and
measurements were made at a scan rate of 100 mV s
21
. Square
wave voltammograms (SWV) were recorded at 20 Hz, E = 5
mV and an amplitude of 10 mV at different conditioning
potentials between 20.80 and 0.50 V vs. SCE.
Next, microaliquots of the corresponding nucleic acid
(tRNA, ssDNA and dsDNA) stock solutions were added to
This journal is The Royal Society of Chemistry 2002
DOI: 10.1039/b111548k Analyst, 2002, 127, 329332 329
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obtain 20 mgL
21
in cell; in the case of dGMP, a concentration
in the order of millimoles per litre was achieved. Cyclic and
square wave voltammograms were registered in conditions
analogous to those with the blank only, so the background
current could be subtracted if desired.
All electrochemical experiments were performed using a
computer controlled m-AUTOLAB potentiostat (Eco-Chemie,
Utrecht, Netherlands), with a standard three electrode system
consisting of a BDD film working electrode, a spiral-wound
platinum wire as counter electrode and a saturated calomel
electrode, SCE (Radiometer, Copenhagen), as reference elec-
trode respectively. Experiments were performed at 20 2 C.
Results
Fig. 1 shows voltammograms recorded at 100 mV s
21
at a BDD
electrode for dGMP, transfer RNA, single and double stranded
DNA on the first scan between 20.80 and 1.50 V after
background correction. Current at potentials more negative than
0.80 V (vs. SCE) coincide with that for the blank only and are
therefore not shown. Two peaks are observed at more positive
potentials and the peak potentials are shown in Table 1.
According to previous work by Dryhurst et al.
26
these can both
be directly attributed to the oxidation of the guanosine groups,
as has been found on mercury and carbon electrodes.
7,2730
It
was found that whilst dGMP and tRNA gave a reproducible
curve in the first and all subsequent experiments, oxidation of
single stranded DNA was followed by a complete dis-
appearance of the signal after the first scan. This was attributed
to surface passivation. In these latter cases, in subsequent work
the electrode was cleaned before every experiment by introduc-
ing it into a saturated solution of sodium hydroxide in ethanol
and then rinsing with ultrapure sterilised water. Using this
procedure reproducible first scans were always obtained.
Double stranded DNA gave a signal comparable to that
observed with the single stranded molecule. Its oxidation was
also followed by surface passivation but was successfully
restored using the procedure described above for single stranded
DNA.
SWV was performed at 20 Hz, E = 5 mV, giving a scan
rate of 100 mV s
21
analogous to that for CV, and an amplitude
of 10 mV; the background corrected scans are shown in Fig. 2.
In all cases well resolved peaks were observed. The more
positive peak was subsequently used for quantification.
7
In order to optimise the response, different pre-conditioning
potentials and pre-oxidation times were investigated. Using
various conditioning potentials between 20.80 and 0.50 V for
each of these compounds in turn, it was found that the
voltammetric signals, both cyclic and square wave, remained
essentially constant. This behaviour is entirely different to that
found on other electrodic materials based on carbon which show
in the positive region an important increase in the electro-
chemical response due to adsorptive behaviour.
The effect of the conditioning time on each compound was
investigated between 10 and 600 s. tRNA showed a behaviour
independent of the preconditioning procedure and an essentially
constant signal was obtained. A linear relationship between
cyclic voltammetry peak height and v
1/2
suggesting a diffusion-
controlled oxidation was observed. For both single and double
stranded DNA the square wave voltammetry oxidation signal
Fig. 1 Background subtracted cyclic voltammograms after 300s at 20.80 V. (a) dGMP 0.5 mM, (b) tRNA 20 mg L
21
, (c) ssDNA 20 mg L
21
, (d) dsDNA
20 mg L
21
. Scan rate 100 mV s
21
.
Table 1 Peak potentials referred to SCE for the different species
studies
First
oxidation
peak
a
Second
oxidation
peak
a
First
oxidation
peak
b
Second
oxidation
peak
b
dGMP 1.15 1.35 1.10 1.30
tRNA 1.10 1.35 1.10 1.35
ssDNA 1.10 1.35 1.05 1.34
dsDNA 1.10 1.35 1.05 1.34
a
Cyclic voltammetry.
b
Square wave voltammetry.
330 Analyst, 2002, 127, 329332
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Fig. 2 Background subtracted square wave voltammograms after 300 s at 20.80 V. (a) dGMP 0.5 mM, (b) tRNA 20 mgL
21
, (c) ssDNA 20 mg L
21
, (d)
dsDNA 20 mg L
21
. Frequency 20 Hz, E = 5 mV and amplitude 10 mV.
Fig. 3 Intensity of cyclic voltammetry peak at 1.10 V versus v
1/2
for (a) dGMP 0.1 mM, (b) tRNA 20 mg L
21
, (b) ssDNA 20 mg L
21
and (d) dsDNA 20
mg L
21
. Linear relationships suggest that the electro-oxidation process is controlled by diffusion.
Analyst, 2002, 127, 329332 331
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was also found to be independent of pre-conditioning time or
potential, and both gave a similar signal regardless of whether
the electro-active group, guanosine, was embedded into the
double helix structure in the double stranded molecule or not.
The cyclic voltammograms of these two compounds also show
a linear relationship with the square root of the scan rate. In the
case of dGMP 0.5 mM it was again observed that the signal was
largely independent of the pre-conditioning time, but a small
increment of 10% was found when comparing the longer (600
s) with respect to the shorter times (10 s). This suggests that the
electro-oxidation of dGMP is essentially controlled by diffu-
sion, although partially coupled to weak adsorption.
Fig. 3 shows that for all four substances studied the
voltammetric peak heights vary linearly with v
1/2
and not versus
v, as would be the case if the process was controlled by
adsorption. The slope of the plots provides information about
the diffusion coefficients, D: assuming a 2-electron-oxidation
mechanism
20
a diffusion coefficient of 1.2 3 10
26
cm
2
s
21
for
dGMP was obtained but a value of 1.2 3 10
25
cm
2
s
21
results
if the mechanism only involves one electron. An estimation of
the diffusion coefficient using the Wilke-Chang semi-empirical
relationship
31
predicts a value of 4.5 3 10
26
cm
2
s
21
, closer to
the 1.5 electron-mechanism proposed by Dryhurst
26
for oxida-
tion on pyrolytic graphite electrode for dGMP. In the case of the
high molecular weight substances the value for the diffusion
coefficient was not directly obtainable because the exact
number of electrons involved was unknown. However, in order
to prove consistency it was taken into account that the number
of active groups, guanine, comprises about 20% of the 50 Kb
that form the Sigma calf thymus DNA used, and from the
diffusion coefficient value obtained by Bard et al.,
32
it was
reasonably inferred that only 10% of these groups react (2
electrons each) with the BDD electrode surface.
A plot of logarithm of peak current versus T
21
showed an
activation energy of 9.5 K Jmol
21
, consistent with the range of
the diffusion activation energies in aqueous solutions
33
(the
LSV technique is sensitive to D
1/2
so that the gradient of the plot
is related to half the activation energy for diffusion).
The independence of the oxidation signal on time or potential
for single and double stranded DNA samples on BDD suggests
that no adsorption processes are involved, in complete contrast
to what is observed on other carbon-based electrodes. This BDD
offers the prospect of simple diffusion controlled voltammetry
for the analytical detection of these molecules.
Conclusions
Highly conductive boron-doped diamond electrodes facilitate
the electrochemical detection of nucleic acids, including double
stranded DNA, in aqueous solution with the advantages of solid
electrodes using classical amperometric methods. The well-
defined peaks observed in SWV are directly assignable to the
electro-oxidation of guanosine groups in the nucleic acid
molecules.
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