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Further development of an electrochemical DNA hybridization biosensor based on long-range electron transfer Elicia L.S. Wong, Freya J. Mearns, Justin J. Gooding ∗ School of Chemistry, The University of New South Wales, Sydney, NSW 2052, Australia Available online 28 April 2005
Abstract An electrochemical DNA hybridization biosensor which exploits long-range electron transfer through double-stranded DNA (ds-DNA) to a redox intercalator is described. The DNA recognition interface consisted of a mixed self-assembled monolayer of synthetic thiolated single-stranded DNA (ss-DNA) and 6-mercapto-1-hexanol (MCH). The target DNA detection is performed electrochemically through cyclic and Osteryoung square wave voltammetry, using anthraquinone derivatives as the intercalators. This biosensor has the ability to differentiate complementary target ss-DNA from non-complementary target, and most importantly, it is able to detect single-base mismatch target ss-DNA through diminution in voltammetric current. The viability of this biosensor has also been investigated through selectivity studies in the presence of interferences and the generality of the detection scheme. © 2005 Elsevier B.V. All rights reserved. Keywords: Long-range electron transfer; DNA hybridization biosensor; Intercalator; Anthraquinone
1. Introduction The development of DNA hybridization biosensors is motivated by applications to wide-scale genetic testing, clinical diagnostics, fast detection of biological warfare agents and environmental testing. Optical , electrochemical [2–6] and microgravimetrical  DNA transductions have been widely studied. The electrochemical DNA biosensor is, among all, able to offer a simple, rapid yet accurate, low-cost pointof-care detection of selected DNA sequences and this has been the topic of considerable interest to many researchers in recent times [8–11]. Different forms of electrochemical DNA biosensors have been developed which transduce DNA hybridization using either a redox active molecule or a label free [12–17] method which relies either on the intrinsic redox-active properties of DNA bases or a change of electrical properties of an electrode interface upon hybridization. Typical label-free transduction methods rely on monitoring the oxidation current of the most electroactive DNA bases, guanine. In one example, Wang et al. immobilized inosine∗
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substituted probe single-stranded DNA (ss-DNA) onto a carbon paste electrode and used direct chronopotentiometry to detect hybridization by monitoring the appearance of a peak due to the oxidation of guanine of the target DNA . As oxidation of guanine bases occurs at a highly positive potential (∼1.0 V versus Ag/AgCl), DNA detection via the oxidation of guanine is associated with high background signals, which compromises the sensitivity of the biosensor. Label methods rely on using a redox-active molecule that has redox activity at low potential and has different binding affinities towards the ss-DNA and ds-DNA. There are numerous labeled electrochemical DNA biosensors and the label can be an enzyme , DNA groove binder [5,19,20] or intercalator [2,3,21]. In a typical assay, the probe ss-DNA is immobilized on a transducer and is subsequently exposed to the target DNA. In most cases, a current is observed prior to hybridization and hence hybridization is transduced by a change in the magnitude of the electrochemical current [4,5]. For example, in the work of Millan and Mikkelsen the label is a cationic redox-active molecule, tris(1,10-phenanthroline) cobalt(III) perchlorate Co(phen)3 3+ . As DNA is anionic, the reporter molecule is attracted to the DNA-modified interface, where it adsorbs and gives a redox current. A greater
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electrochemical current is observed when hybridization occurs due to the increased attraction of the Co(phen)3 3+ to the DNA which is partly because of the greater anionic character of ds-DNA on the transducer surface. Intercalators as redox labels for detecting DNA hybridization are particularly interesting because they interact with the “-stack” formed by the DNA-base pairs in the DNA duplex. With some intercalators efficient electron transfer over long distances between the intercalator and the electrode can proceed via the DNA duplex but not with a single strand of DNA. The utilization of long-range electron transfer as the basis of a DNA hybridization biosensor was first introduced by Barton and co-workers , where electrochemical current was observed through DNA duplexes by using methylene blue (MB) as an intercalator. Any disruptions in the DNA-base pairing affect the perfect -stacking and causes attenuation in the electrochemical current which enables the detection of single-base mismatches without requiring stringency washes. The method relied on a close packed array of DNA duplexes on the electrode surface to ensure that no MB could access the electrode directly and thus allow the differentiation between current due to the reduction of MB via long-range electron transfer and reduction current due to direct access of MB to the electrode surface. Recently the idea of using long-range electron transfer to transduce DNA hybridization has been extended using an anionic intercalator, 2, 6-anthraquinone disulphonic acid (AQDS) . Thiolated probe ss-DNA was immobilized onto the gold electrode using the facile gold-sulfur self-assembly chemistry. As the bases of DNA have affinity to the gold surfaces, a diluent layer of 6-mercapto-1-hexanol was then added to the probe ss-DNA modified surface for two purposes: (i) to project the ss-DNA into the solution to ease hybridization and (ii) to eliminate any non-specific interaction from either target DNA or the redox-active molecule. This DNA recognition interface was exposed to target complementary DNA and after formation of DNA duplex, the ds-DNA modified electrode was incubated in an AQDS solution. The AQDS was able to intercalate into the DNA duplex, typically at the distal end of the DNA, several nanometers from the electrode. AQDS as the redox intercalator proved to be remarkably successful, as it gives no electrochemical current prior to hybridization (that is no current is detected if only ss-DNA are present). This absence of electrochemical current is due to the electrostatic repulsion of anionic AQDS molecules away from the DNA interface due to the alcohol terminus of the MCH. As a result, any AQDS current observed after exposure to complementary target could be attributed to long-range electron transfer with the DNA acting as a conduit for the electron transfer. This approach allows not only the transduction of DNA hybridization but also discrimination of both C–A mismatches and G–A mismatches without further amplification . There were a number of aspects of this initial study with AQDS, however, which required further investigation. Points of interest included how to reduce the assay time, whether
the DNA-modified interface could detect the target sequence of DNA in a cocktail of many different sequences of DNA, whether the direction of the sequence or the base sequence composition influenced the performance of the biosensor and whether other anthraquinone-based redox intercalators possessed the ability to detect G–A as well as C–A mismatches. The purpose of this paper is to answer these questions. The intercalators used are the anthraquinone derivatives, AQDS which was employed in the initial work, and 2-anthraquinone monosulphonic acid (AQMS), which was previously reported as intercalating into the DNA more rapidly than AQDS .
2. Experimental 2.1. Apparatus All electrochemical measurements were performed with a BAS 100B electrochemical analyser (Bioanalytical Systems Inc., Lafayette, IN, USA). Voltammetric experiments were carried out with a conventional three-electrode system, consisting of a bare or modified gold working electrode, a platinum flag counter electrode and an Ag/AgCl/3.0 M NaCl reference electrode (Bioanalytical Systems Inc., Lafayette, IN, USA). All potentials are reported versus Ag/AgCl reference at room temperature. The electrochemical measuring solution was degassed with argon for approximately 15 min prior to data acquisition and was blanketed under an argon atmosphere during the entire experimental period. Cyclic voltammetry (CV) was conducted at a sweep rate of 100 mV s−1 . Osteryoung square wave voltammetry (OSWV) was conducted at a pulse amplitude of 25 mV, a step of 4 mV and a frequency of 10 Hz. 2.2. Reagents 6-Mercapto-1-hexanol (MCH, 97% purity), anthraquinone-2-sulfonic acid, sodium salt monohydrate (AQMS, 97% purity), and anthraquinone-2,6-disulfonic acid, disodium salt (AQDS, 97% purity) were purchased from Aldrich Chemicals (Sydney, Australia). Calf thymus DNA, salmon testes DNA and salmon sperm DNA were purchased from Sigma (Sydney, Australia). Reagent-grade K2 HPO4 , KH2 PO4 , NaCl, KCl, NaOH, HCl and absolute ethanol were purchased from Ajax Chemicals Pty. Ltd. (Sydney, Australia). All reagents were used without further purification. All solutions were prepared using Milli-Q water. The 20mer deoxyoligonucleotides were purchased from Proligo Pty. Ltd. (Sydney, Australia). The base sequences are as follows: Thiolated p53 DNA probe (20-base sequence DNA 1a): 5 -GGGGCAGTGCCTCACAACCT-p-(CH2 )3 -SH-3 Thiolated Barr–Epstein DNA probe (20-base sequence DNA 1b): 5 -SH-(CH2 )6 -p-AGGGATGCCTGGACACAAGA-3
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Fig. 1. Schematic fabrication of the DNA recognition interface for the unbound intercalator system and subsequent transduction of a hybridization event. The thiolated probe DNA is self-assembled onto the gold electrode via the facile gold-thiol chemistry followed by self-assembly of an MCH diluent layer. This recognition interface is exposed to target complementary DNA and upon hybridization the ds-DNA/MCH modified electrode is then immersed in the intercalator solution for intercalation to occur.
Complementary DNA target to DNA 1a (20-base sequence DNA 2a): 5 -AGGTTGTGAGGCACTGCCCC-3 Complementary DNA target to DNA 1b (20-base sequence DNA 2b): 5 -TCTTGTGTCCAGGCATCCCT-3 C–A mismatch DNA target to DNA 1a (20-base sequence DNA 3): 5 -AGGTTGTGAGGCCCTGCCCC-3 G–A mismatch DNA target DNA 1a (20-base sequence DNA 4): 5 -AGGTTGTGAGGCGCTGCCCC-3 Non-complementary DNA target (20-base sequence DNA 5): 5 -GGATGGACGAAGCGCTCAGG-3 All oligonucleotide stock solutions were prepared with 10 mM Tris–HCl, (pH 8.00) and stored in a −80 ◦ C freezer until use. Calf thymus, salmon testes and salmon sperm DNAes were denatured before use. 2.3. Preparation of buffer solutions Immobilization buffer contained 1 M KH2 PO4 (pH 4.5); hybridization buffer contained 10 mM Tris–HCl, 1 M NaCl (pH 7.0) and phosphate buffer, in which electrochemical experiments were performed, contained 0.05 M K2 HPO4 /KH2 PO4 , 0.3 M NaCl (pH 7.0). The 1 mM AQDS and 1 mM AQMS stock solutions both contained 0.2 M KCl and 0.05 M KH2 PO4 /K2 HPO4 (pH 7.0). The pH was adjusted with either NaOH or HCl solution. Milli-Q water and all buffers were sterilized using an autoclave.
2.4. Procedures—preparation of probe ss-DNA and subsequent transduction of hybridization The DNA recognition interface was prepared as described previously  and as shown in Fig. 1. Working gold electrodes were constructed and prepared as described previously . Briefly, a clean gold electrode was incubated in 4 M thiolated ss-DNA 1 at room temperature for 90 min, followed by adsorption of MCH monolayer in 1 mM ethanolic solution for 30 min. This recognition interface was then exposed to the target oligomer 2, 3, 4 or 5, immersed into 4 M solutions of target DNA in hybridization buffer for 150 min, followed by rinsing with phosphate buffer. Detection of hybridization was performed by immersing the ds-DNA covered surface in intercalator solution for 3 to 6 h (intercalator dependent) for intercalation into the duplexes to occur. Electrochemical detection of hybridization was performed through subsequent voltammetric experiments (either CV or OSWV) in 0.05 M K2 HPO4 / KH2 PO4 , containing 0.3 M NaCl (pH 7.0).
3. Results and discussion To determine whether hybridization via long-range electron transfer could be achieved, the probe DNA-modified electrode was incubated in 1 mM AQDS for 6 h, rinsed in copious amounts of water and then placed in an AQDS-free solution for electrochemical measurement. The CVs and OSWVs (Fig. 2) obtained were featureless with no redox peaks
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transfer through the DNA. The AQDS biosensor has also been used to probe single-base C–A and G–A mismatches. There is approximately 15% (for C–A) and 34% (for G–A) of the voltammetric current observed with the complementary target. The diminution in AQDS current observed in both C–A and G–A mismatches is a result of the base-pair disruptions, which perturb the -base stacking of the DNA duplex. G–A mismatches are known to be thermodynamically more stable than C–A mismatches , and therefore, G–A pairing does not perturb the DNA -stacking as significantly as C–A pairing which is consistent with the greater current diminution observed for the C–A mismatch as compared to the G–A mismatch. Viability of this biosensor has been further investigated through selectivity studies and generality of the detection scheme. 3.1. Selectivity of the AQDS biosensor Fig. 2. OSWVs of the DNA recognition interface prior to (a) and after exposure to complementary target ss-DNA (b), single-base G–A mismatch target ss-DNA (c), single-base C–A mismatch target ss-DNA (d) and noncomplementary target ss-DNA (e). Peak currents were obtained from OSWVs in 0.05 M phosphate buffer, 0.3 M NaCl (pH 7.0) at step of 4 mV, pulse amplitude of 25 mV and frequency of 10 Hz after immersion in 1 mM AQDS solution for 6.5 h, followed by rinsing with 0.05 M phosphate buffer. Flat OSWV was obtained prior to exposure to target DNA as the anionic AQDS molecules are effectively screened off by the negative-dipole of the MCH diluent layer. Prominent currents due to long-range electron transfer were obtained after exposure to complementary, single-base G–A and C–A mismatches target DNA. There were seven- and three-fold decreases in observed current for C–A and G–A mismatches when they were compared with the observed current for a perfectly match target sequence.
around −450 mV versus Ag/AgCl as expected for AQDS. However, after exposure to complementary target DNA, followed by 6-h incubation in AQDS, a prominent AQDS current was observed in both CV and OSWV. The change in current shows transduction of DNA hybridization, which can be achieved with the current attributed to long-range electron
Selectivity studies of the DNA biosensor based on longrange electron transfer were performed by immersion of the DNA recognition interface in a cocktail of DNA which contains equal amounts of complementary target and a noncomplementary target. The non-complementary targets include 4 M denatured calf thymus, salmon testes, salmon sperm and 20-mer synthetic ss-DNA. As usual, electrochemical currents were only observed after exposure to the DNA cocktail (Fig. 3a). The current density obtained for the biosensor upon exposure to pure 4 M of complementary target was 14.0 ± 1.5 A cm−2 (n = 10). After exposure to mixed 4 M of complementary target, the current densities obtained were 12.8, 13.6, 12.4 and 13.2 A cm−2 (Fig. 3b) in the presence of salmon testes, salmon sperm, calf thymus and 20-mer synthetic ss-DNA, respectively. This shows that the DNA biosensor using AQDS is able to select the complementary target in the presence of other DNA species without any significant attenuation in current. Furthermore, the absence of a
Fig. 3. (a) CV of the DNA recognition interface prior to (a) and after (b) exposure to 4 M complementary target and 4 M calf thymus DNA. All voltammetric experiment were performed in 0.05 M phosphate buffer, 0.3 M NaCl (pH 7.0) after incubation in 1 mM AQDS for 6.5 h and rinsing with 0.05 M phosphate buffer. The scan rate was 100 mV s−1 . (b) Bar chart showing the current density obtained upon exposure to cocktail of target DNA, only slight decrease in current density is observed for the DNA cocktail showing the high selectivity of the AQDS biosensor towards its complementary target.
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change in double-layer capacitance for the complementary DNA detected in a cocktail of other DNA (66.2 F cm−2 ), relative to when only the complementary DNA is present (64.1 F cm−2 ), shows that the alcohol-terminated diluent layer resists non-specific adsorption of DNA. If the DNA non-specifically adsorbed, the access of ions to the electrode surface would be restricted by the DNA; thus the capacitance would decrease. Two other important issues were whether the long-range electron transfer approach was only specific to 20-mer synthetic thiolated probe ss-DNA 1a or whether it is generic and applicable to other short genome sequences and if the immobilization from the 3 end or 5 end of the DNA had an influence on the biosensor performance. Probe ss-DNA 1a is part of the p53 gene that codes for a protein for tumour suppression . We tested the generality of this detection scheme using another probe DNA, 20-mer synthetic thiolated probe ss-DNA 1b, which is part of the Barr Epstein gene that is responsible for the herpes virus (Epstein–Barr Virus). Using probe ss-DNA 1b, no electrochemical currents were observed in both CV and OSWV prior to hybridization and currents were only observed after exposure to complementary target (Fig. 4). The current density obtained for probe ss-DNA 1b biosensor was 4.81 A cm−2 , which is identical to that observed with the sequence in 1a when the same length thiol linker between the electrode and the DNA was used (Fig. 4). This suggests that the orientation of the probe, whether it is immobilized from 3 end or 5 end of the DNA, has no effect on the analytical performance of the biosensor. The one drawback of using AQDS for DNA biosensing via long-range electron transfer was the long assay time required. This is due to a slow AQDS intercalation rate caused by electrostatic repulsion between AQDS and the DNA du-
plexes. Further study on this detection scheme is aimed at reducing the assay time without compromising the absence of a background signal prior to hybridization and the ability to discriminate between a perfect complement and targets with single base pair mismatches.
The strategy that has been investigated to reduce the overall assay time is by using a less anionic intercalator than AQDS to increase the rate of intercalation of the redox molecule without compromising the absence of an electrochemical signal from unhybridized DNA on the electrode surface. The best intercalator identified so far is 2anthraquinone-monosulphonic acid (AQMS), which took half the time to intercalate, 3 h versus 6 h . This more rapid interaction time enabled the overall assay time to be decreased from 8.5 h to 6.5 h, but the previous study did not reveal whether the ability to discriminate single base pair mismatches was compromised at all. The ability of the AQMS biosensor to detect C–A and G–A mismatches was determined by immersing the recognition interface into target DNA 2a (the complementary sequence), 3 (a C–A mismatch) and 4 (a G–A mismatch). The OSWVs upon exposure to complementary, non-complementary and both mismatched target ss-DNA are shown in Fig. 5. All the mismatches occur in the middle of the DNA strand. There is approximately 10% for C–A and 30% for G–A of the voltammetric current observed for the complementary target. The ability of AQMS to detect both C–A and G–A mismatches is consistent with an AQDS biosensor where similar percentages of diminution in voltammetric currents are observed.
Fig. 4. OSWVs of the probe ss-DNA 1b recognition interface prior to (a) and after hybridization with complementary target (b), and with the sequence in probe ss-DNA 1a after hybridization (c) when the same carbon length thiol linker as probe ss-DNA 1b was used (C6). Experimental conditions were as described in the caption for Fig. 2.
Fig. 5. OSWVs of the AQMS DNA recognition interface after exposure to complementary target ss-DNA (a), single-base G–A mismatch target ss-DNA (b), single-base C–A mismatch target ss-DNA (c) and noncomplementary target ss-DNA (d). Experimental conditions were as described in the caption for Fig. 2.
3.2. Long-range electron transfer via AQMS biosensor
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Characterization of the DNA-modified interface electrochemically as described previously  showed the number of immobilized probe ss-DNA to be 16 ± 4 pmol cm−2 (n = 10) by chronocoulometry (results not shown), which is consistent with that found electrochemically  and via neutron scattering . After hybridization with the complementary target sequence there was, on average, one AQMS molecules intercalated per DNA strand with a hybridization efficiency of ∼ 85%. The rate of electron transfer (kET ) is 3.0 s−1 using the Laviron theory , which is twice that calculated for when AQDS was used (1.5 s−1 ) in the biosensor. This suggested that AQMS molecules are better coupled within the ds-DNA than AQDS molecules. AQMS has the clear advantage over AQDS in reducing the assay time and the kET suggested that AQMS is better coupled into the ds-DNA. While complete absence of current is obtained for AQDS biosensor prior to hybridization, the non-specific adsorption of AQMS can also be completely eliminated and hence is not an issue. Therefore, we have successfully found another anionic intercalator, which is able to transduce hybridization via long-range electron transfer with shorter assay time.
4. Conclusion Following our initial investigation on AQDS biosensors, which are able to detect DNA mismatches, we have further studied the viability of the AQDS by selectivity studies and the generality of the detection scheme. It was found that the AQDS biosensors are selective to the complementary target even in the presence of salmon testes, salmon sperm, calf thymus or 20-bases non-complementary synthetic target DNA. The detection scheme is also generic as it is able to detect Barr–Epstein complementary sequence. The use of another anionic intercalator, AQMS, which is less negatively charged than AQDS, further shortened the assay time without compromising the advantages using long-range electron transfer for transducing DNA hybridization.
Acknowledgements We would like to thank the Australian Research Council (ARC) for the funding of this project. F.J.M. would like to acknowledge the Australian Institute of Nuclear Science and Engineering (AINSE) for her postgraduate stipend.
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Biographies Elicia L.S. Wong received her BSc in anatomy, DipGrad in mathematics and MSc in bioorganic chemistry from the University of Otago, New Zealand. She is currently studying towards her PhD under the supervision
of Dr J. Justin Gooding at the University of New South Wales, Australia. The main focus of her current research is on development of biosensors for the detection of nucleic acids and small molecules which have affinity to DNA. Freya J. Mearns completed a BTech in forensic and analytical chemistry in 1999 and BSc (Hons) in 2000 at the Flinders University of South Australia, Australia. She is currently a PhD student at the University of New South Wales, Australia, and is researching DNA hybridization biosensors. J. Justin Gooding is a senior lecturer and the leader of biosensor research at The University of New South Wales. He obtained a DPhil from Oxford University under the guidance of Prof Richard Compton before becoming a post-doctoral research associate at the Institute of Biotechnology at Cambridge University. In 1997 he returned to his native Australia as a Vice-Chancellor Post-Doctoral Research Fellow at the University of New South Wales before taking up an academic position in 1998.