1. INSTITUTE OF PHYSICS PUBLISHING MEASUREMENT SCIENCE AND TECHNOLOGY
Meas. Sci. Technol. 15 (2004) R1–R11 PII: S0957-0233(04)56971-0
REVIEW ARTICLE
Recent trends in electrochemical DNA
biosensor technology
Kagan Kerman, Masaaki Kobayashi and Eiichi Tamiya1
School of Materials Science, Japan Advanced Institute of Science and Technology,
1-1 Asahidai, Tatsunokuchi, Ishikawa 923-1292, Japan
E-mail: tamiya@jaist.ac.jp
Received 2 October 2003, accepted for publication 7 November 2003
Published 11 December 2003
Online at stacks.iop.org/MST/15/R1 (DOI: 10.1088/0957-0233/15/2/R01)
Abstract
Recent trends and challenges in the electrochemical methods for the
detection of DNA hybridization are reviewed. Electrochemistry has superior
properties over the other existing measurement systems, because
electrochemical biosensors can provide rapid, simple and low-cost on-field
detection. Electrochemical measurement protocols are also suitable for
mass fabrication of miniaturized devices. Electrochemical detection of
hybridization is mainly based on the differences in the electrochemical
behaviour of the labels towards the hybridization reaction on the electrode
surface or in the solution. Basic criteria for electrochemical DNA biosensor
technology, and already commercialized products, are also introduced.
Future prospects towards PCR-free DNA chips are discussed.
Keywords: electrochemical DNA hybridization biosensor, transduction of
DNA hybridization, DNA recognition interface, redox-labels, label-free
detection, metal nanoparticles, electrical detection
Introduction Electrochemical detection of hybridization is mainly
based on the differences in the electrochemical behaviour of
Since the new concept of ‘the electrochemical DNA the labels with or without double-stranded DNA (dsDNA) or
hybridization biosensor’ was first introduced by Millan and single-stranded DNA (ssDNA). The labels for hybridization
Mikkelsen back in 1993 [1], this exciting research area has detection can be anticancer agents, organic dyes, metal
received intense attention from several groups around the complexes, enzymes or metal nanoparticles. There are
world. DNA biosensors convert the Watson–Crick base pair basically four different pathways for electrochemical detection
recognition event into a readable analytical signal. A basic of DNA hybridization:
DNA biosensor is designed by the immobilization of a single- (1) A decrease/increase in the oxidation/reduction peak
stranded (ss) oligonucleotide (probe) on a transducer surface current of the label, which selectively binds with
to recognize its complementary (target) DNA sequence via dsDNA/ssDNA, is monitored.
hybridization. The DNA duplex formed on the electrode (2) A decrease/increase in the oxidation/reduction peak
surface is known as a hybrid. This event is then converted current of electroactive DNA bases such as guanine or
into an analytical signal by the transducer, which can adenine is monitored.
be an electrochemical [2], optical [3], gravimetric [4], (3) The electrochemical signal of the substrate after
surface plasmon resonance-based [5] or electrical [6] device. hybridization with an enzyme-tagged probe is monitored.
Electrochemistry has superior properties over the other existing (4) The electrochemical signal of a metal nanoparticle probe
measurement systems, because electrochemical biosensors attached after hybridization with the target is monitored.
enable fast, simple and low-cost detection.
Recently, various reviews of electrochemical DNA
1 Author to whom any correspondence should be addressed.
biosensors have been reported [7–9]. The present review
0957-0233/04/020001+11$30.00 © 2004 IOP Publishing Ltd Printed in the UK R1

2. Review Article
A DNA biosensor for the detection of hepatitis B virus
(HBV) was developed by covalently immobilizing ss HBV
DNA fragments to a gold (Au) electrode surface via a
carboxylate ester to link the 3 -hydroxy end of the DNA to the
carboxyl of the thioglycolic acid monolayer [24]. A 181 bp
HBV DNA fragment of known sequence was obtained and
amplified by the polymerase chain reaction (PCR). The surface
hybridization of the immobilized HBV probe with its target
DNA fragment was detected by using the electrochemical
signal of osmium bipyridine. The formation of the hybrid
on the Au electrode resulted in a great increase in the
peak current of osmium bipyridine in comparison with those
obtained at a bare or probe-modified electrode. The difference
between the responses of ferrocenium hexafluorophosphate at
the probe- and hybrid-modified Au electrodes suggested that
an electrochemical hybridization biosensor could be used to
monitor DNA hybridization [25].
An electrochemical hybridization assay was designed by
Yamashita et al [26] for the rapid analysis of a heterozygous
Scheme 1. Label-based electrochemical detection of DNA deficiency related to the human lipoprotein lipase (LPL) gene.
hybridization. An intercalator, which can selectively bind to the
PCR-amplified samples containing the wild-type sequence, a
hybrid on the electrode surface, causes an increase in the
electrochemical signal. At the probe-modified electrode, a lower mutant or a one-base deletion of the LPL gene were subjected
signal than the one obtained from the hybrid modified electrode is to hybridization with a 13–15 mer DNA probe that represented
monitored, because the intercalator cannot accumulate on the either the wild-type or the mutated sequence immobilized on
surface. an Au electrode. The differential pulse voltammetry of the
electrode before and after hybridization was determined in the
will focus on the most recent developments in the technology presence of a bis-intercalator, namely ferrocenylnaphthalene
of electrochemical DNA biosensors, while providing basic diimide (FND), at 460 mV.
insight for the detection methods. An overview of the current Electrochemical detection of several types of one-base
status of DNA biosensor development will include information mismatch in a 20 mer hybrid anchored on a gold electrode
on applications and future prospects for molecular diagnostics. was also performed by Yamashita et al [27] in connection with
FND as the label.
A probe-modified Au electrode was incubated in 2,6-
1. Label-based electrochemical detection of DNA
disulfonic acid anthraquinone (AQDS) intercalator solution,
hybridization
rinsed and placed in an AQDS-free buffer solution, whereupon
1.1. Redox-active molecules as labels for hybridization voltammetric experiments were performed [28]. No
detection voltammetric peaks were observed for probe-modified Au
electrodes. Upon DNA hybridization and incubation in AQDS,
The electrochemical detection of DNA hybridization based on clear voltammetric peaks were observed. The absence of
a redox-active label is illustrated in scheme 1. Basically, the an AQDS signal for probe-modified surfaces clearly showed
hybrid modified electrode is immersed in a solution which that the electrochemistry is due to long-range electron transfer
contains a redox-active and DNA-binding molecule. After through the DNA duplex.
a period of time for the interaction between the DNA and Since the redox-active molecule-based detection of
the molecule, an electrochemical technique is applied to the hybridization is well established, two commercialized DNA
electrode to measure the surface species. If the redox-active chips are now being introduced onto the molecular diagnosis
molecule is an intercalator such as daunomycin [10], it would market. The first example of such a DNA chip, called the
be inserted between the double-helix structure of the dsDNA eSensorTM , was produced by Motorola Life Sciences Inc. [29].
with the help of its planar aromatic ring, and an enhancement Electrochemical detection by using the eSensorTM ,
in the redox signal would be observed. On the contrary, if the which is illustrated in scheme 2, proceeds via a sandwich
molecule had an affinity towards ssDNA, such as methylene hybridization assay, in which three critical components
blue [11–13], then a high signal would be observed from the (capture probe, target and signalling probe) are each present
probe-modified electrode. These changes in the peak potential in the device. The signalling probe, tagged with a ferrocene,
current of the labels for the probe and hybrid molecules provide serves to label the target upon hybridization. Electrons flow
the basis for detection of the label-based hybridization. to the electrode surface only when the target is present,
Several metal complexes such as cobalt phenanthro- and are specifically hybridized to both the signalling and
line [14, 15], cobalt bipyridine [1, 2] and ruthenium bipyri- capture probes [30]. eSensorTM bioelectric chips also
dine [16, 17], anticancer agents such as echinomycin [18, 19] successfully detected 86% of the HPV types contained in
and epirubicin [20], and organic dyes such as methylene clinical samples [31].
blue [21–23] were used as labels for the detection of hybridiza- Toshiba’s electrochemical DNA hybridization detection
tion. system is called the GenelyzerTM [32]. It contains an
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Scheme 2. Electrochemical detection protocol of eSensorTM DNA chips, produced by Motorola Life Sciences Inc. The capture probe,
which is immobilized on the Au electrode, hybridizes with the target DNA. Then, the ferrocene-tagged signalling probe is exposed to the
hybrid on the electrode surface. The redox signal of the tag is measured after the sandwich hybridization between the signalling probe and
target DNA (Fc: ferrocene tag).
electrochemical DNA chip that is able to analyse and type
single-nucleotide polymorphisms (SNPs) and common DNA
sequence variations by using the redox-active dye Hoechst
33258 [33–35]. This hand-held device is only 45 cm wide,
50 cm deep and 23 cm in height. A typical analysis of
SNPs in a sample takes only about 1 hour. A disposable
peptide nucleic acid (PNA) array with a 20-channel electrode
was microfabricated by Hashimoto and Ishimori [36] for the
detection of the cancer gene c-Ki-ras from PCR-amplified
Scheme 3. Enzyme-tagged electrochemical detection of DNA
samples by using Hoechst 33258. hybridization. Specific hybridization between the surface-anchored
A new electrochemical hybridization detection method target and the enzyme-tagged signalling probe enables monitoring
based on Hoechst 33258 has recently been reported by of the amplified voltammetric or amperometric signal from the
Kobayashi et al [37]. Linear sweep and cyclic voltammetry product as the substrate is introduced to the electrode (E: enzyme).
methods were employed on a microchip composed of 32 Au
electrodes, for the detection of target DNA related to the human labels is illustrated in scheme 3. When the substrate is intro-
immunodeficiency virus (HIV) and hepatitis C virus (HCV). duced to the enzyme-modified electrode surface, the electro-
Moreover, a novel electrochemical method for detecting the chemical activity of the product greatly simplifies the detection
hepatitis B virus (HBV) has been reported [38]. Current of DNA hybridization.
methods need a compulsory step, i.e. immobilization of the A 38-base DNA sequence has been detected at a
probe DNA on the electrodes, which adds to the complexity concentration of 20 pmol l−1 in 15–35 µl droplets by means of
of making a DNA chip. However, this novel method, based an electrochemical enzyme-amplified sandwich-type assay on
on DNA aggregation by an electroactive indicator, can detect a mass-manufacturable screen-printed carbon electrode [43].
DNA quantification without applying a compulsory step: the The formation of the sandwich brought the horseradish
immobilization of probe DNA. Furthermore, our new method peroxidase (HRP) label of the detection sequence into
using Hoechst 33258 can detect DNA amplification without electrical contact with a pre-electrodeposited redox polymer,
the DNA purification step. This novel method has enabled us making the sandwich an electrocatalyst for the reduction of
to detect HBV DNA from human blood in less than 2 h. hydrogen peroxide to water at +0.2 V (Ag/AgCl). Recently,
DNA damage was detected by using catalytic oxidation Zhang et al [44] reported a major advance in the horseradish
with ruthenium bipyridine and by monitoring the binding of peroxidase-amplified amperometric detection of DNA hybri-
cobalt bipyridine to DNA [39]. Damaged DNA reacted more dization. They achieved the detection of DNA as low as 3000
rapidly than intact dsDNA with ruthenium bipyridine, giving copies in a 10 µl droplet at a concentration of 0.5 fM.
electrochemical peaks at approximately 1 V that grew larger Pividori et al [45] implemented a classical dot–blot format
with reaction time. Cobalt bipyridine bound more strongly to for the enzyme-based amperometric detection of hybridization.
intact dsDNA, and its signals at 0.04 V, decreased as DNA was The analytical procedure consisted of five steps: DNA
damaged. target immobilization by adsorption onto a nylon membrane;
hybridization between a DNA target and biotin–DNA probe;
complexation reaction between a biotin–DNA probe and HRP-
1.2. Enzyme labelling for detection of DNA hybridization
streptavidin conjugate; integration of the modified membrane
Labelling of probes with enzymes has also been effectively onto an electrochemical transducer; and, finally, amperometric
used for the electrochemical detection of DNA hybridiza- detection using a suitable substrate for the enzyme-labelled
tion [40–42]. The basic detection scheme of using enzymes as duplex.
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Scheme 4. Metal-nanoparticle-based electrochemical detection of DNA hybridization. (A) After the specific hybridization between the
surface-anchored target and the metal-nanoparticle-tagged signalling probe, the intrinsic electrochemical signal of the metal nanoparticle
can be observed on the same electrode. (B) Dissolution of the metal tag with acid treatment enables the detection of hybridization on a
separate bare electrode. (C) Au nanoparticles can be coated with an Ag layer and the electrochemical signal of Ag can be detected with or
without dissolving the Ag layer (Me: metal nanoparticle).
Hairpin-forming probes in connection with HRP were Cholinesterase- and peroxidase-immobilized screen-
used for the electrochemical detection of factor V Leiden printed electrodes were also used to detect the DNA–antibody
mutations from human blood specimens [46]. After adducts [50].
hybridization between the ss target DNA and the fluorescein- Glucose oxidase was also utilized for the detection of viral
labelled detector probe, the hybrids were immobilized on a genes on an Au electrode [51]. The generation of a redox-
carbon paste electrode (CPE). HRP-linked anti-fluorescein active replica was accomplished by using ferrocene conjugated
antibody–enzyme conjugates were then loaded onto each uracil bases in combination with polymerase. The resulting
hybrid. Following the addition of an HRP substrate replica on the electrode surface acted as an electron-transfer
and mediators such as tetramethylbenzidine, amperometic relay for the bioelectrocatalysis of glucose.
signals provided quantitative information on the number of A major breakthrough in DNA biosensors was achieved
immobilized hybrids on the sensor surface. by eliminating the PCR step from DNA diagnosis. This PCR-
Magnetic beads have provided easy removal of non- free biosensor based on enzyme amplification was reported
specifically bound DNA from electrode surfaces [47–49]. An by Patolsky et al [52]. In the presence of polymerase,
enzyme-linked sandwich hybridization assay was taken with a a biotinylated nucleotide, complementary to the mutation
magnetic-particle-labelled probe hybridizing to a biotinylated site, was coupled to the ds hybrid on the electrode surface.
DNA target that captured a streptavidin-alkaline phosphatase Subsequent binding of avidin-alkaline phosphatase to the
assembly, and the biocatalysed precipitation of an insoluble
(AP). The 1-naphthol product of the enzymatic reaction was
product on the surface, provided the lower limit of sensitivity
quantified through its low-potential (+0.1 V versus Ag/AgCl)
of 1 × 10−14 mol ml−1 for Tay–Sachs genetic disorder with no
differential pulse voltammetric peak at the disposable screen-
PCR preamplification.
printed electrode [48].
Palecek et al [49] developed a new technology in
which DNA hybridization was performed on commercially 1.3. DNA biosensors meet nanotechnology
available magnetic beads and detected on solid electrodes. 1.3.1. Metal nanoparticles as labels for detection of
Target DNA was modified with osmium tetroxide, 2, 2 - DNA hybridization. The basic protocol for the detection
bipyridine (Os, bipy) and the immunogenic DNA–Os, bipy of metal-nanoparticle-based DNA hybridization is shown
adduct was determined by the AP-linked immunoassay in scheme 4. There are currently three strategies for
with electrochemical detection. Electro-inactive 1-naphthyl detection: the intrinsic electrochemical signal of the metal
phosphate was used as a substrate, and the electroactive product nanoparticle can be observed with (scheme 4(B)) [53] or
(1-naphthol) was measured on the carbon electrodes. without (scheme 4(A)) [54] dissolving it with acid treatment
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and can be detected on a bare electrode, or the Au nanoparticles internally with ferrocenecarboxyaldehyde marker molecules
can be coated with a silver (Ag) layer to enhance the for amplified electrochemical DNA sensing. A DNA array
electrochemical signal of Ag (scheme 4(C)) [55]. microchip utilizing magnetic beads as labels to detect DNA
Ozsoz et al [54] reported a DNA biosensor based on a hybridization has recently been reported by Miller et al [64].
pencil graphite electrode and modified with the target DNA. Polyvinyl-alcohol-based magnetic beads for rapid and efficient
When the target DNA hybridized with a complementary probe separation, purification and detection of DNA were reported
conjugated to an Au nanoparticle, the Au oxide wave was by Oster et al [65].
monitored to detect the hybridization.
The detection of DNA hybridization using cadmium 1.3.2. Carbon nanotubes for electrochemical detection of DNA
sulfide nanocluster-based electrochemical stripping was hybridization. A nanoelectrode array based on vertically
reported by Zhu et al [56]. Their protocol consisted of the aligned multi-walled carbon nanotubes (MWNTs) embedded
hybridization of the target DNA with the CdS nanocluster in SiO2 was reported by Li et al [66]. Oligonucleotide
oligonucleotide DNA probe, followed by dissolution of the probes were selectively functionalized to the open ends of
CdS nanoclusters anchored on the hybrids and the indirect the MWNTs. The hybridization of subattomole DNA targets
determination of the dissolved cadmium ions by sensitive could be detected by combining the nanoelectrode array with
anodic stripping voltammetry (ASV) at a mercury-coated ruthenium bipyridine mediated guanine oxidation.
glassy-carbon electrode. Carbon-nanotube-based electrochemical assay for hy-
Another assay relied on the hybridization of the target bridization detection provided enhanced daunomycin signals
DNA with a silver nanoparticle oligonucleotide DNA probe, due to the large surface area and good charge-transport char-
followed by the release of the silver metal atoms anchored acteristics of the MWNTs [67].
on the hybrid by oxidative metal dissolution and the indirect
determination of the solubilized Ag(I) ions by ASV at a carbon 1.3.3. Encoding technology with metal nanoparticles and
fibre ultramicroelectrode [57]. enzymes. The labelling of probes bearing different DNA
Wang et al [58] reported the hybridization of a target sequences with different metal nanoparticles or enzymes
oligonucleotide to magnetic bead-linked oligonucleotide enables the simultaneous detection of more than one target
probes followed by binding of the streptavidin-coated metal in a sample as shown in scheme 5. The key point in this
nanoparticles to the captured DNA. After the dissolution of protocol is that the signals of labels should not overlap with
the Au tag, potentiometric stripping measurements of the each other, otherwise simultaneous monitoring of signals in
dissolved Au were performed at single-use thick-film carbon one scan cannot be performed.
electrodes. Three encoding nanoparticles (zinc sulfide, cadmium
An electrochemical DNA detection method was devel- sulfide and lead sulfide) were used to differentiate the signals of
oped for the sensitive quantification of an amplified 406- three DNA targets in connection with stripping-voltammetric
base pair human cytomegalovirus DNA sequence (HCMV measurements of the heavy metal dissolution products [68].
DNA) [59]. The assay relied on These products yielded well-defined and resolved stripping
peaks at −1.12 V (Zn), −0.68 V (Cd) and −0.53 V (Pb) at the
(a) the hybridization of the target HCMV DNA with Au mercury-coated glassy-carbon electrode (versus the Ag/AgCl
nanoparticle-modified oligonucleotide, reference). The position and size of these peaks reflected the
(b) followed by the release of Au by acid treatment, and identity and level of the corresponding DNA target.
(c) the indirect determination of the solubilized AuIII ions Two encoding enzymes, alkaline phosphatase and β-
by ASV at a sandwich-type screen-printed microband galactosidase, were used to differentiate the signals of
electrode. two DNA targets from the signals of their electroactive
products [69]. These products provided well-defined and
Sato et al [60] reported a novel aggregation phenomenon
resolved peaks at +0.31 V (α-naphthol) and +0.63 V (phenol)
of DNA-modified Au nanoparticles induced by hybridization
at the graphite working electrode. The position and size of
of target DNA, which did not crosslink the nanoparticles. Ag-
these peaks reflected the identity and level of the corresponding
gregation of DNA-functionalized poly(isopropylacrylamide)
target.
(PNIPAAm) nanoparticles without the crosslinking mech-
Recently, encoded redox beads, based on the encapsula-
anism was reported by Mori and Maeda [61]. Probe tion of different quantum dots (QD) within polystyrene mi-
oligonucleotide was grafted onto PNIPAAm, which produced crospheres, and encoded redox rods, prepared by sequential
nanoparticles above 40 ◦ C. When the target DNA was perfectly plating of different metal tracers into the pores of a host mem-
complementary to the probe, the nanoparticles aggregated to- brane, have also been designed for electrochemical authenticity
gether at high salt concentration. testing of commercial products [70].
Wang et al [62] recently reported two particle-based
procedures for monitoring DNA hybridization. The first
protocol involved detecting the iron content of magnetic 2. Label-free electrochemical detection of DNA
beads, while the second route relied on probes labelled with hybridization
Au-coated iron core-shell nanoparticles. In both protocols,
2.1. Electrical charge flow through DNA for detection of
iron-containing particles were dissolved, and the released
hybridization
iron was quantified by cathodic-stripping voltammetry in the
presence of the 1-nitroso-2-naphtol ligand and a bromate Intense debate has occurred on the question of whether or
catalyst. Wang et al [63] loaded microsphere tags not DNA is able to conduct electrical charges. Fluorescence
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Scheme 5. Encoding technology enables simultaneous detection of several DNA targets in one measurement. Different metal nanoparticles
or enzymes can be used as the signalling probes for coding several targets simultaneously (E: enzyme, M: metal nanoparticle, S: substrate,
P: product; the subscript numbers represent three different types).
quenching experiments in connection with intercalated donor Silver deposition facilitated by the nanoparticles bridged the
and acceptor molecules on DNA have generated contradictory microgap and led to a change in conductivity.
discussions. The conductivity of DNA has been assessed from The flow of electrical charges through DNA provided the
electron transfer as a function of the distance between the donor basis for the detection of DNA hybridization. One of the
and acceptor molecules [71]. leading DNA chip venture companies, GeneOhm Sciences
The first electrical transport measurements on DNA Inc., and its technology for detecting single-nucleotide
molecules at least 600 nm long were reported by Fink and polymorphisms (SNPs), uses DNA’s ability to carry electrical
Sch¨ nenberger [72]. Direct measurements of electrical current
o current [77].
as a function of the potential applied across DNA indicated A probe, which has a DNA sequence complementary
efficient conduction through the DNA ‘ropes’. The resistivity to a portion (15–30 bases) of a gene with an SNP, is
values derived from these measurements were comparable to immobilized onto an Au electrode surface as a self-assembled
those of conducting polymers; thus DNA was assumed to monolayer (SAM). After hybridization with the target DNA,
transport electrical current as efficiently as a semiconductor. the electrode is exposed to an intercalator solution. As the
Large numbers of DNA molecules aligned on films also intercalator accumulates between the base pairs of the hybrid,
showed anisotropic conductivity [73]. In contrast, electrical a current is applied to the hybrid from the electrode. If the
hybrid is perfectly matched, then the current flow reaches the
transport through individual poly(G)–poly(C) DNA molecules
intercalator and a high charge signal can be obtained. If there
10.4 nm long connected to two metal nanoelectrodes indicated
is an SNP in the hybrid, the flow of current is blocked and
large-bandgap semiconducting behaviour [74]. Non-linear
no charge signal can be monitored from the DNA SAM. A
current–voltage curves that exhibited a voltage gap at low
schematic representation of the core technology of GeneOhm
applied bias were obtained. A peak structure, which exhibited
Sciences Inc. is shown in scheme 6.
the voltage dependence of the differential conductance,
A comparison of electron transfer rates of ferrocenoyl-
suggested that charge carrier transport was mediated by the linked DNA molecules has recently been reported by Long
molecular energy bands of DNA [74]. et al [78]. Boon et al [79] reported the development of an
Braun et al [75] hybridized target DNA with surface- electrochemical assay for protein binding to DNA-modified
bound oligonucletides on two Au electrodes, thus stretching electrodes based on the detection of associated perturbations in
the DNA molecule in the microgap between these electrodes. DNA base stacking. Au electrode surfaces that were modified
DNA was then used as the template for the vectoral growth of with loosely packed DNA duplexes, covalently crosslinked to
a conductive silver wire. a redox-active intercalator and containing the binding site of
Array-based electrical detection of DNA with Au- the test protein, were constructed. Charge transport through
nanoparticle-tagged probes was reported by Park et al [76]. DNA as a function of protein binding was then assayed.
Probe oligonucleotides were immobilized into a 20 µm gap Marques et al [80] used electrochemical techniques
between Au and Ti microelectrodes. Hybridization between to detect DNA polymorphisms in human genes by using
the probes and the Au-nanoparticle-tagged complementary cytochrome P450 3A4 (CYP3A4) as a model gene. Their
DNA localized the Au nanoparticles in the microgap. detection protocol was based on dsDNA’s ability to transport
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Scheme 6. Electrochemical detection protocol of electrical DNA chips, produced by GeneOhm Sciences Inc. After hybridization of the
probe with the target DNA, the hybrid-modified Au electrode is exposed to an intercalator solution. Then a current is applied to the hybrid
from the electrode. If the hybrid is perfectly matched, the current flow reaches the intercalator and a high charge signal can be obtained. A
mismatch in the hybrid blocks the flow of current, so no charge signal can be monitored from the hybrid.
charge along nucleotide stacking. The perturbation of the
double-helix pi-stack introduced by a mismatched nucleotide
reduced electron flow, and could be detected by measuring the
attenuation of the charge transfer. CYP3A4*1A homozygotes
could be detected by 5 µC charge attenuation.
The reagentless transduction of DNA hybridization into
a readily detectable electrochemical signal by means of a
conformational change approach was reported by Fan et al
[81]. The strategy involved an electroactive, ferrocene-tagged
DNA stem-loop structure that self-assembled onto an Au
electrode by means of facile Au-thiol chemistry. Hybridization Scheme 7. Label-free electrochemical detection of DNA
induced a large conformational change in this surface-confined hybridization eliminates the use of redox molecules, and shortens
DNA structure, which in turn significantly altered the electron- the assay time. The inosine (I) substituted probe shows no
electrochemical signals, since inosine is electro-inactive. After
transfer tunnelling distance between the electrode and the hybridization with the target DNA, the appearance of the guanine
redoxable label. The resulting change in electron-transfer (G) oxidation signal provides specific detection.
efficiency was readily measured by cyclic voltammetry at
target DNA concentrations as low as 10 pM.
a new detection method for DNA hybridization as shown in
A new label-free electrochemical DNA hybridization
scheme 7. This procedure eliminated the use of external labels
detection method has recently been reported by Gooding et al
and shortened the assay time. Inosine-substituted probes for
[82]. The change in flexibility of an ss probe before and after
DNA hybridization biosensors were applied onto indium tin
hybridization, caused an ion-gating effect, where the rigid rod-
oxide electrodes by Thorp [89]. Jelen et al [90] showed that
like structure of the duplex opened up the SAM-modified Au
electrode to the access of ions. subnanomolar concentrations (related to monomer content)
of unlabelled DNA could be determined using copper solid
amalgam electrodes or hanging mercury drop electrodes in the
2.2. Intrinsic DNA signals for the detection of hybridization presence of copper ions.
Guanine and adenine are the most electroactive bases of Strepavidin and biotin are widely used in affinity-based
DNA, because they can easily be adsorbed and oxidized on separations and diagnostic assays. Recently, strepavidin
carbon electrodes. Guanine and adenine oxidation signals on and avidin were electrochemically detected in solution by
carbon electrodes can be observed at around 1.0 and 1.3 V in adsorptive transfer-stripping voltammetry at a CPE [91].
0.50 M acetate buffer solution (pH 4.80, ABS), respectively, Especially, an avidin-modified CPE could successfully bind
as reported by Jelen et al [83]. Monitoring the changes in biotin-modified oligonucleotides onto the electrode surface.
these signals upon duplex formation enabled the detection of This avidin–biotin binding technology was applied for the
hybridization [84–88]. The electrochemical signals obtained detection of transgenic avidin maize.
from free adenine and guanine bases decreased on binding Toxicity controls of wastewater samples were also
to their complementary thymine and cytosine bases after conducted by using DNA biosensors [92]. The DNA biosensor
hybridization. was assembled by immobilizing dsDNA on the surface of
The use of inosine-substituted probes and the appearance a disposable, carbon screen-printed electrode (SPE). The
of a guanine signal upon hybridization with the target enabled oxidation signal of guanine was used as the analytical signal.
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The presence of compounds with an affinity for DNA was oligonucleotides onto silica surfaces and their subsequent hy-
measured by their effect on the guanine oxidation. A bridization with complementary strands [99]. The impedance
comparison of the results with a toxicity test based on measurements, which provided a direct means of detecting
bioluminescent bacteria confirmed the applicability of the variations in electrical charge accumulation across the semi-
method to real samples. conductor/oxide/electrolyte structure when the oxide surface
The interaction of small molecules with DNA was was chemically modified, showed that the semiconductor’s
detected by monitoring the changes in the adenine and flat band potential underwent reproducible shifts of −150 and
guanine signals. The interaction of an alkylating agent, 4, 4 - −100 mV following the immobilization and the hybridization
dihydroxy chalcone, with dsDNA and ssDNA was studied steps, respectively.
electrochemically based on the oxidation signals of guanine An electrochemical method to detect DNA hybridization
and adenine by using differential pulse voltammetry (DPV) at directly was developed on the basis of a new conductive
a CPE [93]. polymer, which was polymerized on a glassy-carbon electrode
The interaction of arsenic trioxide (As2 O3 ) with dsDNA, with a terthiophene monomer having the carboxyl group
ssDNA and also 17 mer short oligonucleotide was studied (3 -carboxyl-5, 2 , 5 , 2 -terthiophene) [100]. A difference in
electrochemically by using DPV with a CPE at the surface admittance was observed before and after hybridization as a
and also in solution [94]. Potentiometric stripping analysis result of the reduction of the resistance after hybridization.
(PSA) was employed to monitor the interaction of As2 O3 with The highest difference in admittance was observed around
dsDNA in solution phase by using a renewable pencil graphite 1 kHz before and after hybridization. Hybridization amounts
electrode. of end two-base and centre one-base mismatched sequences
An investigation of the niclosamide–DNA interaction were obtained only in a 14.3% response when compared to
using an electrochemical DNA biosensor showed for the first that for the complementary matched sequence.
time clear evidence of interaction with DNA and suggested The use of electrochemical impedance spectroscopy (EIS)
that niclosamide toxicity can be caused by this interaction, and the conducting polymer, poly(pyrrole), as an integrated
after reductive activation [95]. recognition and transduction system for reagentless biosensor
The interaction of rhodium dimers, including the carboxy- systems was demonstrated for two different systems by Farace
lates (acetate, propionate, butyrate, trifluoroacetate, citrate and et al [101]. A construct for discriminating DNA hybridization
gluconate), amidates (acetamidate and trifluoroacetamidate) and able to differentiate ssDNA and dsDNA based on the
and carboxamidate (Doyle catalyst S), with DNA was investi- interaction of the DNA with poly(pyrrole) was achieved.
gated by electrochemical methods [96]. DPV measurements
showed that most of the rhodium carboxylates had a higher 2.4. Field-effect sensors for the detection of DNA
affinity for adenine than guanine residues. hybridization
Microfabricated silicon field-effect sensors were used to
2.3. Electrochemical impedance for the detection of DNA directly monitor the increase in surface charge when DNA
hybridization was hybridized on the sensor surface [102]. The electrostatic
Hybridization of two complementary DNA strands on the immobilization of probe DNA on a positively charged poly-l-
surface of the structure induced a variation in the flat band lysine layer allowed hybridization at low ionic strength, where
potential of the semiconductor leading to a shift of impedance field-effect sensing was the most sensitive case. Nanomolar
curves along the potential axis. This means that it is possible to DNA concentrations could be detected within minutes, and a
detect DNA hybridization directly without the use of labelled single-base mismatch within 12 mer oligonucleotides could be
probes by monitoring impedance [97]. distinguished by using a differential detection technique with
Alkaline phosphatase oxidative hydrolysis of the soluble two sensors in parallel.
5-bromo-4-chloro-3-indoyl phosphate to the insoluble indigo
product was utilized by Patolsky et al [98] in two different 3. Future prospects
sensing configurations. The accumulation of the insoluble
product on Au electrodes or Au/quartz crystals changed the As the three major electrochemical DNA chips produced
electron-transfer resistance at the electrode surface or the mass by GeneOhm Sciences Inc., Toshiba Corp. and Motorola
associated with the piezoelectric crystal, thus enabling the Life Sciences Inc. enter the molecular diagnosis market in
quantitative transduction of DNA hybridization by Faraday the near future, it is likely that the competition and interest
impedance spectroscopy or microgravimetric quartz-crystal in the electrochemical detection of DNA hybridization will
microbalance measurements, respectively [98]. intensify. Although the main target in all the DNA chip
The capacitance measurement, electrochemical impedance technologies has been to eliminate the role of the polymerase
spectroscopy and constant current chronopotentiometry have chain reaction (PCR) from their protocols, this goal has
been used for the electrochemical study of echinomycin and not yet been reached by commercialized electrochemical
its interaction with ssDNA and dsDNA at the hanging mercury technologies. Some efforts are now being made towards
drop electrode (HMDE) [18]. Echinomycin was found to be decreasing the time needed for PCR. The only promising
a promising redox indicator for hybridization detection due to example for the achievement of a PCR-free DNA biosensor
its strong binding ability with dsDNA. based on the enzymatic amplification of electrochemical
Radiolabelling and electrochemical impedance measure- signals was reported by Patolsky et al [52]. With the rapid
ments were used to characterize the immobilization of ss homo- progress in the electrochemical biosensing world, it can be
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9. Review Article
envisaged that DNA biosensors without PCR amplification [15] Erdem A, Meric B, Kerman K, Dalbasti T and Ozsoz M 1999
will soon be on the market. Therefore, electrochemical Detection of interaction between metal complex indicator
DNA biosensors with their cost-effectiveness and suitability and DNA by using electrochemical biosensor
Electroanalysis 11 1372–6
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popular in the near future. Eckhardt A E and Thorp H H 1997 Probing biomolecule
recognition with electron transfer: electrochemical sensors
for DNA hybridization Bioconjug. Chem. 8 906–13
Acknowledgments [17] Yang I V, Ropp P A and Thorp H H 2002 Toward
electrochemical resolution of two genes on one electrode:
KK acknowledges the Monbukagakusho scholarship for using 7-deaza analogues of guanine and adenine to prepare
research students from the Japan Ministry of Education, PCR products with differential redox activity Anal. Chem.
Culture, Sports, Science and Technology (MEXT). The authors 74 347–54
express their gratitude to Dr Yasutaka Morita for a critical [18] Hason S, Dvorak J, Jelen F and Vetterl V 2002 Interaction of
reading of the manuscript. DNA with echinomycin at the mercury electrode surface
as detected by impedance and chronopotentiometric
measurements Talanta 56 905–13
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