Electrochemical analysis of nucleic acids at boron-doped diamond electrodes

César Prado a, Gerd-Uwe Flechsig b, Peter Gründler b, John S. Foord a, Frank Marken c and Richard G. Compton *a
aPhysical and Theoretical Chemistry Laboratory, University of Oxford, South Parks Road, Oxford, UK OX1 3QZ. E-mail: compton@ermine.ox.ac.uk
bUniversität Rostock, FB Chemie, Technische und Umweltchemie, Abt. für Analytische, Albert Einstein Str. 3a, D-18051 , Rostock, Germany
cDepartment of Chemistry, Loughborough University, Loughborough, UK LE11 3TU

Received 18th December 2001 , Accepted 14th January 2002

First published on 29th January 2002


Highly conductive boron-doped diamond (BDD) electrodes are well suited for performing electrochemical measurements 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 involving thermal heating or ultrasonic treatment. tRNA, single and double stranded DNA and 2′-deoxyguanosine 5′-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 1960’s1,2 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 voltammetry3–7 and chronopotentiometric adsorptive stripping voltammetry (CPSA)8–12 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 chronopotentiometric adsorptive detection on carbon paste and pyrolytic graphite electrodes8,9,14–18 has been successfully applied to the study of nucleic acids with various results. Recently the use of heated carbon paste electrodes19,20 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 electrodes21,22 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.22–24 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. 2′-deoxyguanosine 5′-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−1 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−1 were prepared in water. Solutions were prepared using UHQ grade water, of resistivity not less than 18 MΩ 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 × 5 × 0.535 mm3, film resistivity 0.1 Ω) 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−1. Square wave voltammograms (SWV) were recorded at 20 Hz, △E = 5 mV and an amplitude of 10 mV at different conditioning potentials between −0.80 and 0.50 V vs. SCE.

Next, microaliquots of the corresponding nucleic acid (tRNA, ssDNA and dsDNA) stock solutions were added to obtain 20 mgL−1 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 μ-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 electrode respectively. Experiments were performed at 20 ± 2 °C.

Results

Fig. 1 shows voltammograms recorded at 100 mV s−1 at a BDD electrode for dGMP, transfer RNA, single and double stranded DNA on the first scan between −0.80 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.7,27–30 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 disappearance 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 introducing 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.
Background subtracted cyclic voltammograms after 300s at −0.80 V. (a) dGMP 0.5 mM, (b) tRNA 20 mg L−1, (c) ssDNA 20 mg L−1, (d) dsDNA 20 mg L−1. Scan rate 100 mV s−1.
Fig. 1 Background subtracted cyclic voltammograms after 300s at −0.80 V. (a) dGMP 0.5 mM, (b) tRNA 20 mg L−1, (c) ssDNA 20 mg L−1, (d) dsDNA 20 mg L−1. Scan rate 100 mV s−1.
Table 1 Peak potentials referred to SCE for the different species studies
  First oxidation peaka Second oxidation peaka First oxidation peakb Second oxidation peakb
a Cyclic voltammetry. b Square wave voltammetry.
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


SWV was performed at 20 Hz, △E = 5 mV, giving a scan rate of 100 mV s−1 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


Background subtracted square wave voltammograms after 300 s at −0.80 V. (a) dGMP 0.5 mM, (b) tRNA 20 mgL−1, (c) ssDNA 20 mg L−1, (d) dsDNA 20 mg L−1. Frequency 20 Hz, △E = 5 mV and amplitude 10 mV.
Fig. 2 Background subtracted square wave voltammograms after 300 s at −0.80 V. (a) dGMP 0.5 mM, (b) tRNA 20 mgL−1, (c) ssDNA 20 mg L−1, (d) dsDNA 20 mg L−1. Frequency 20 Hz, △E = 5 mV and amplitude 10 mV.

In order to optimise the response, different pre-conditioning potentials and pre-oxidation times were investigated. Using various conditioning potentials between −0.80 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 electrochemical 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 v1/2 suggesting a diffusion-controlled oxidation was observed. For both single and double stranded DNA the square wave voltammetry oxidation signal 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 diffusion, although partially coupled to weak adsorption.

Fig. 3 shows that for all four substances studied the voltammetric peak heights vary linearly with v1/2 and not versusv, 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 mechanism20 a diffusion coefficient of 1.2 × 10−6 cm2 s−1 for dGMP was obtained but a value of 1.2 × 10−5 cm2 s−1 results if the mechanism only involves one electron. An estimation of the diffusion coefficient using the Wilke-Chang semi-empirical relationship31 predicts a value of 4.5 × 10−6 cm2 s−1, closer to the 1.5 electron-mechanism proposed by Dryhurst26 for oxidation 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.


Intensity of cyclic voltammetry peak at 1.10 V versusv1/2 for (a) dGMP 0.1 mM, (b) tRNA 20 mg L−1, (b) ssDNA 20 mg L−1 and (d) dsDNA 20 mg L−1. Linear relationships suggest that the electro-oxidation process is controlled by diffusion.
Fig. 3 Intensity of cyclic voltammetry peak at 1.10 V versusv1/2 for (a) dGMP 0.1 mM, (b) tRNA 20 mg L−1, (b) ssDNA 20 mg L−1 and (d) dsDNA 20 mg L−1. Linear relationships suggest that the electro-oxidation process is controlled by diffusion.

A plot of logarithm of peak current versusT−1 showed an ‘activation energy’ of 9.5 K Jmol−1, consistent with the range of the diffusion activation energies in aqueous solutions33 (the LSV technique is sensitive to D1/2 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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