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Review
Electrochemical nucleic acid biosensors
Joseph Wang∗
Department of Chemistry and Biochemistry, College of Arts and Sciences, New Mexico State University,
Box 30001-Dept. 3C, Las Cruces, NM 88003, USA
Received 13 June 2001; received in revised form 5 September 2001; accepted 10 September 2001
Abstract
Electrochemical devices have received considerable attention in the development of sequence-specific DNA hybridization
biosensors. Such devices rely on the conversion of the DNA base-pair recognition event into a useful electrical signal. Electro-
chemical biosensing of DNA hybridization is not only uniquely qualified for meeting the size, cost, and power requirements
of decentralized genetic testing, but offer an elegant route for interfacing—at the molecular level—the DNA-recognition and
signal-transduction elements. This article reviews current directions in electrochemical DNA biosensors, and discusses recent
strategies and future prospects for such electrical detection.
© 2002 Elsevier Science B.V. All rights reserved.
Keywords: DNA biosensors; Electrochemistry; Voltammetry; Electrode
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 1 ) 0 1 3 9 9 - X
64 J. Wang / Analytica Chimica Acta 469 (2002) 63–71
interactions thus offers great promise for developing resulting from the Watson–Crick base-pair recog-
simple, rapid, and user-friendly DNA sensing devices nition event, under controlled potential conditions
(in a manner analogous to miniaturized blood-glucose [2,4,5]. The probe-coated electrode is commonly
meters). Recent efforts have led to a host of new immersed into a solution of a target DNA whose
strategies for electrical detection of DNA hybridiza- nucleotide sequence is to be tested. When the target
tion [2,4,5]. Such electrochemical avenues for gener- DNA contains a sequence which matches that of the
ating the hybridization signal are the subject of the immobilized oligonucleotide probe DNA, a hybrid du-
present review. plex DNA is formed at the electrode surface (Fig. 1).
Such hybridization event is commonly detected via
the increased current signal of an electroactive indi-
2. Electrochemical biosensing cator (that preferentially binds to the DNA duplex),
of DNA hybridization in connection to the use of enzyme labels or redox
labels, or from other hybridization-induced changes
Electrochemical detection of DNA hybridization in electrochemical parameters (e.g. capacitance or
usually involves monitoring of a current response, conductivity).
Fig. 1. Major processes involved in electrochemical DNA biosensors based on the use of redox indicators (Ox).
J. Wang / Analytica Chimica Acta 469 (2002) 63–71 65
attention to experimental variables affecting the hy- [14]. Such electronic control has been used also for
bridization event at the transducer-solution interface. differentiating among oligonucleotide hybrids. Me-
These include the salt concentration, temperature, vis- chanically renewed electrodes, including polishable
cosity, the presence of accelerating agents, contacting biocomposites and graphite pencils, have also been
time, base composition (G + C, %), and length of used for regenerating a ‘fresh’ probe layer [15,16].
probe sequence. Careful control of the hybridization Alternately, one can use “one-shot” screen-printed
event is thus required. The stability of hybrids formed electrodes, similar to those used for self-testing of
between strands with mismatched bases is decreased blood glucose, and hence obviate the need for regen-
according to the number and location of the mis- eration [11]. Such disposable DNA sensor strips also
matches. Many DNA biosensors are not capable of meet the needs of many decentralized genetic testing.
selectively detecting a point mutation, as desired in
numerous practical situations. Controlling the strin- 2.3. Electrochemical transduction
gency of hybridization, particularly using elevated of DNA hybridization
temperatures, can thus be used for discriminating
among oligonucleotide hybrids (including mismatch The hybridization event is commonly detected via
discrimination). Control of the hybridization time can the increased current signal of a redox indicator (that
be used for tuning the linear dynamic range, with associates with the newly formed surface hybrid), or
shorter time offering an extended range at the cost of from changes in electrochemical parameters (such as
lower sensitivity [12]. Detection limits ranging from capacitance or conductivity) or in the redox activity of
the nanomolar to the picomolar concentration range the nucleic acid resulting from the duplex formation.
can thus be achieved in connection to 5 and 60 min
hybridization times. Even lower detection limits can 2.3.1. Indicator-based detection
be attained in connection to advanced amplification Earlier devices have relied primarily on the use
protocols (described in the following sections). of electroactive hybridization indicators [4]. Such
We have demonstrated that significantly enhanced indicators are small electroactive DNA-intercalating
selectivity can be achieved by the use of peptide nu- or groove-binding substances, that posses a much
cleic acid (PNA) probes [13]. Such DNA analogues, higher affinity for the resulting hybrid compared
possess an uncharged pseudopeptide backbone (in- to the single-stranded probe. Accordingly, the con-
stead of the charged phosphate-sugar one of natu- centration of the indicator at the electrode surface
ral DNA). Because of their neutral backbone, PNA increases when hybridization occurs, resulting in in-
probes offer greater affinity in binding to comple- creased electrochemical response. Besides effective
mentary DNA, and improved distinction between differentiation between ss- and ds-DNA, the indicator
closely related sequences (including the detection of should possess a well-defined, low-potential, voltam-
single-base imperfections). This is attributed to the fact metric response. Such properties of redox indicators
that mismatch in PNA/DNA duplexes is much more are essential for attaining high sensitivity and selec-
destabilizing than in a DNA/DNA duplexes (with a tivity. Both linear-scan or square-wave voltammetric
lowering of the Tm by 15 versus 11 ◦ C, respectively). modes [17] or constant-current chronopotentiometry
Such mismatch discrimination is of particular impor- [18] can be used to detect the association of the redox
tance in the detection of disease-related mutations. indicator with the surface duplex.
Proper attention should be given also to the Mikkelsen’s group, that pioneered the use of redox
reusability of the DNA biosensors, namely to the indicators, demonstrated its utility for detecting the
regeneration of the surface-bound single-stranded cystic fibrosis F508 deletion sequence associated
probe after each assay. Both thermal and chemical with 70% of cystic fibrosis patients [19]. A detection
(sodium hydroxide, urea) regeneration schemes have limit of 1.8 fmol was demonstrated for the 4000-base
been shown useful for ‘removing’ the bound target DNA fragment in connection to a Co(bpy)3 3+ marker.
in connection with different DNA biosensor formats. High selectivity towards the disease sequence—but
Even more elegant is the use of controlled electric not to the normal DNA—was achieved by performing
fields for facilitating the denaturation of the duplex the hybridization at an elevated temperature of 43 ◦ C.
J. Wang / Analytica Chimica Acta 469 (2002) 63–71 67
Table 1
Examples of redox-active indicators used for the biosensing of DNA hybridization
Indicator Detection mode transducer Electrode Ep,a vs. Ag/AgCl (V) Reference
event could be achieved in connection to the use of liposomes resulted in a dramatic signal amplification
horseradish-peroxidase (HRP) labeled target and an (of ca. 105 ). The same enzyme label was employed for
electron-conducting redox polymer. In this system, quantitative pulse amperometric monitoring of PCR
the hybridization of enzyme labelled oligo(dA)25 tar- amplification [32] and for differential pulse measure-
get with oligo(dT)25 probe, covalently attached to ments of sequences related to human cytomegalovirus
electron-conducting redox hydrogel resulted in the DNA [33]. The coupling of enzyme-based DNA as-
‘wiring’ of the enzyme to the transducer, and in a con- says with an efficient magnetic removal of unwanted
tinuous hydrogen-peroxide electroreduction current. sample constituents has been illustrated in our labo-
A single-base mismatch in an 18-base oligonucleotide ratory [34]. Enhanced “wiring” of enzyme labels is
was thus detected using a 7 m-diameter carbon-fiber anticipated through the emerging use of nanoparticle
transducer. A HRP label has been combined by Patol- superstructures [35].
sky et al. [30] with a biocatalytic precipitative accu-
mulation of the enzyme-generating product to achieve 2.3.3. Label-free electrochemical biosensing
multiple amplifications and hence, extremely low of DNA hybridization
detection limits. Chronopotentiometry and faradaic Increased attention has been given recently to direct
impedance spectroscopy were employed for detect- label-free electrochemical detection schemes, in which
ing the biocatalyzed deposition reaction. Applica- the hybridization event triggers a change in an electri-
bility for the detection of mutations relevant to the cal signal. Such protocols greatly simplify the sensing
Tay–Sachs genetic disorder was demonstrated. En- protocol (as they eliminate the need for the indicator
hanced amplification of DNA sensing processes was addition/association/detection steps) and offers an in-
achieved also by using liposomes labeled with HRP stantaneous detection of the duplex formation. Such
in connection to faradaic impedance spectroscopic direct, in situ detection can be accomplished by mon-
detection [31] (e.g. Fig. 4). Such use of functionalized itoring changes in the intrinsic redox activity of the
Fig. 4. Amplified electrical detection of DNA hybridization using HRP-functionalized liposomes and biocatalytic precipitation of the
product of the enzymatic reaction (from [31] with permission).
J. Wang / Analytica Chimica Acta 469 (2002) 63–71 69
nucleic acid target or probe or changes in the electro- observed in the presence of the single-base mismatch.
chemical properties of the interface. For example, it is Prospects for designing electronic circuits based on
possible to exploit changes in the intrinsic electroac- manipulation of charge transport through DNA were
tivity of DNA accrued from the hybridization event discussed [44].
[36–39]. Among the four nucleic acids bases, the gua- Direct, label-free, electrical detection of DNA hy-
nine moiety is most easily oxidized and is most suit- bridization has also been accomplished by monitoring
able for such indicator-free hybridization detection. changes in the conductivity of conducting polymer
To overcome the limitations of the probe sequences molecular interfaces, e.g. using DNA-substituted
(absence of G), guanines in the probe sequence were or doped polypyrrole films [45,46]. For example,
substituted by inosine residues (pairing with C’s) and Korri-Youssoufi et al. [45] has demonstrated that
the hybridization was detected through the target DNA a 13-mer oligonucleotide-substituted polypyrrole
guanine signal [36]. A greatly amplified guanine sig- (PPy) film displays a decrease current response
nal, and hence, hybridization response can be obtained during the duplex formation. Such change in the
by using the electrocatalytic action of a Ru(bpy)3 2+ electronic properties of PPy has been attributed to
redox mediator [37]. This involves the following cat- bulky conformational changes along the polymer
alytic cycle: backbone due to its higher rigidity following the
hybridization.
Ru(bpy)3 2+ → Ru(bpy)3 3+ + e− (1)
New avenues for generating the hybridization
Ru(bpy)3 3+ + G → Ru(bpy)3 2+ + G+ (2) signal are currently being explored in several lab-
oratories. Siontorou et al. [47] reported on the use
The presence of a guanine-containing nucleic acid of self-assembled bilayer lipid membranes (BLMs)
target thus creates a catalytic cycle that results in for the direct monitoring of DNA hybridization. A
a large current output. The ability of this approach decrease in the ion conductivity across the lipid mem-
to detect mutations or deletions involving guanine brane surface, containing the single-stranded probe,
bases has been demonstrated [40]. This technology was observed during the formation of the duplex. This
is currently being used in a commercial nucleic acid was attributed to alterations in the ion permeation as-
detection system (of Xanthon Inc.) that uses a 96-well sociated with structural changes in the BLM accrued
microtiter plate format, where each well contains by the desorption of the ds-DNA. The mechanism of
seven separate probe-coated indium–tin oxide elec- interaction between oligonucleotides and BLM films
trodes (two of which are used as controls) [41]. A was examined by Hianik et al [48]. Aoki et al. de-
single microtiter plate thus allows 672 measurements veloped a novel ion-channel protocol for the indirect
(480 tests and 192 controls). In addition to anodic biosensing of DNA hybridization [49]. The system
measurements of the target guanine, it is possible relied on the electrostatic repulsion of the diffusing
to use cathodic stripping measurements of the target ferrocyanide redox marker, accrued from the hy-
adenine for sensitive detection of DNA hybridization bridization of the negatively-charged target DNA and
[39]. the neutral PNA probe (Fig. 5). High specificity to-
It is possible also to exploit different rates of wards mismatch oligonucleotides was demonstrated.
electron transfer through ss- and ds-DNA for prob- A related approach for amplifying DNA hybridiza-
ing hybridization (including mutation detection) via tion signals, based on the use of negatively-charged
the perturbation in charge migration through DNA. liposomes, was described by Patolsky et al. [50].
Barton and coworkers demonstrated that such charge Such liposomes bind to the bound target to form a
transport is disrupted by the presence of a single-base ‘giant’ negatively-charged interface that repels the
mismatch [42,43]. Such disruption and point muta- anionic redox probe. The resulting barrier to the in-
tion were detected, using a gold electrode modified terfacial electron transfer was monitored by Faradaic
with thiolated DNA, by monitoring changes in the impedance spectroscopy.
charge transport between an electroactive methylene Berggren et al. demonstrated that changes in the
blue intercalator and a ferricyanide redox species. capacitance of a thiolated-oligonucleotide modified
A substantially smaller electrocatalytic signal was gold electrode, provoked by hybridization to the
70 J. Wang / Analytica Chimica Acta 469 (2002) 63–71
Fig. 5. An ion-channel sensor based on a PNA probe immobilized on gold electrode, and detection of the hybridization based on the
electrostatic repulsion of a negatively-charged redox marker (shown as an octahedron) (from [49] with permission).
complementary strand (and the corresponding dis- as integration of various processes, including sample
placement of solvent molecules from the surface), can collection, DNA extraction and amplification, with the
be used for monitoring in high sensitivity and speed actual hybridization detection, on a single microchip
the hybridization event [51]. platform containing multiple functional elements and
related microfluidic network. Such integration and
miniaturization should lead to significant advantages
3. Conclusions and outlook in terms of cost, speed, sample/reagent consumption,
simplicity, and automation. The integration of mul-
Over the past decade, we have witnessed a tremen- tiple biosensors in connection to DNA microarrays
dous progress towards the development of electro- should lead to the simultaneous analysis of multiple
chemical nucleic acid biosensors. Such devices are of nucleic acid sequences, and hence, to the generation of
considerable interest due to their tremendous promise characteristics hybridization patterns and acquisition
for obtaining sequence-specific information in a faster, of expression information. Screening of DNA-protein
simpler, and cheaper manner compared to traditional or DNA-drug interactions would also benefit from
nucleic acid assays. In addition to excellent economic such DNA microarrays. The rapid progress that elec-
prospects, such devices offer innovative routes for in- trical detection for DNA hybridization has made in
terfacing (at the molecular level) the DNA-recognition the last decade suggests the major impact it may have
and signal-transduction elements, i.e. an exciting op- in the present decade. It is envisioned that future re-
portunity for fundamental research. The realization search will lead to new electrical detection strategies,
of instant decentralized (medical, environmental, or that coupled with major technological advances, will
forensic) DNA testing would require additional devel- result in powerful, miniaturized, easy-to-use instru-
opmental work. Particular attention should be given ments for DNA diagnostics. Such instruments will
to the major challenges of mismatch discrimination, undoubtedly accelerate the realization of large-scale
signal amplification, non-specific adsorbates, as well genetic testing.
J. Wang / Analytica Chimica Acta 469 (2002) 63–71 71