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An introduction to electrochemical DNA

biosensors
Katherine J. Odenthal and J. Justin Gooding*
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DOI: 10.1039/b701816a

Electrochemical DNA biosensors exploit the affinity of single-stranded DNA for

complementary strands of DNA and are used in the detection of specific
sequences of DNA with a view towards developing portable analytical devices.
Great progress has been made in this field but there are still numerous challenges
to overcome. This review for researchers new to the field describes the
components of electrochemical DNA biosensors and the important issues in their
design. Methods of transducing DNA binding events are discussed along with
future directions for DNA biosensors.

Introduction
The detection of genetic disorders is
clearly of importance for preventative
health care. Many of these disorders are
caused by mutations of the double-
stranded DNA (ds-DNA) in our cells
that comprises the blueprint of our
genetic makeup. Double-stranded DNA
is formed by two single strands (ss-DNA)
bonding to each other to form the well-
known double helix. Because of the way
the two strands wind around each other,
a DNA duplex has two grooves spiralling
around the outside of the DNA duplex.
The larger one is called the major groove
and the smaller one the minor groove, as
shown in Fig. 1. The double helix forms
when all the bases on one strand match
with their partner bases on the opposite
strand. This pairing of bases, known as
WatsonCrick base pairing, sees a gua-
nine base matched with a cytosine and
thymine matched with adenine. Many
disorders are caused by even a single
mismatch in this base pairing. Single
base-pair mismatches are also known as
single nucleotide polymorphisms or
SNPs. Hence the detection of specific
sequences of DNA and the base-pair
composition is important in diagnosing
genetic disorders.
DNA biosensors are commonly
employed to detect specific sequences of
DNA. They can achieve this by exploit-
ing the specific affinity of ss-DNA for a
School of Chemistry, University of New South
Wales, Sydney, NSW 2052, Australia.
E-mail: justin.gooding@unsw.edu.au
Fig. 1 Recognition interface consisting of the immobilised DNA probe and steps in the
detection of a specific DNA target strand.
complementary strand of ss-DNA. DNA
microarrays are one example of a
laboratory-based DNA biosensor that
has found enormous use in scientific
research for gene identification and gene
expression profiling.1 Ideally though, for
the detection of genetic disorders, a
portable device that could be used in a
point of care environment for quick
diagnosis is desired.2 Electrochemical
DNA biosensors are one strategy being
explored for the development of DNA
biosensors as portable devices. This is
because electrochemical technology is
amenable to miniaturisation, can be
accurate and sensitive with simple self-
contained instrumentation and easily
controlled reaction conditions.2
Biosensors consist of two main parts: a
biorecognition interface, that enables the
selective detection of the analyte, and the
transducer, which converts the recogni-
tion event into an electronic signal that
can be outputted to the end user (see
Fig. 1). In electrochemical DNA biosen-
sors, the transducer is an electrode onto
which DNA as the biorecognition species
is immobilised. It follows then that there
are two aspects in developing a DNA
biosensor. Firstly the fabrication of the
biorecognition layer which involves the
immobilisation of DNA. Second is a
method of transducing the biorecogni-
tion event of a single strand of DNA in
solution hybridising with the immobi-
lised strand. There have been two main
approaches to the electrochemical trans-
duction of DNA hybridisation, which
can be broadly referred to as labelled
methods and label-free methods. Labelled
methods use redox active molecules that
bind to DNA. They can do this in several
ways including by binding in the minor
groove (see Fig. 1), intercalating a planar
aromatic ring between base pairs or by
interaction with one of the bases. The

This journal is The Royal Society of Chemistry 2007 Analyst, 2007, 132, 603610 | 603

first electrochemical DNA hybridisation
biosensor was reported by Mikkelsen and
co-workers36 using the minor groove
binding redox active label Co(Phen)33+
that gave different electrochemical signals
depending on whether the DNA was
Published on 23 April 2007. Downloaded by Indian Institute of Technology Chennai on 23/10/2017 15:00:10.
double- or single-stranded. There are also
label-free methods which rely on either
changes to the electrical characteristics of
the DNA-modified interface upon hybri-
disation or on the natural electroactivity
of DNA. Both the label-free and labelled
approaches will be discussed below. Work
in this area has been looking to achieve
both sensitivity and selectivity, and these
are the driving forces behind much of the
research to date.710
The purpose of this paper is to
introduce electrochemical DNA biosen-
sors and to outline the important factors
in designing the biorecognition interface
and different approaches to determining
transduction. It is important to note that
the DNA biosensor is really the end
point of an entire analytical device with
steps related to removing the DNA
from cells, purifying it, cutting it into
usable lengths and amplifying it, all
preceding the actual detection by the
DNA biosensor.
How to design the
biorecognition interface
For a biosensor to detect DNA, it is
necessary to have a biorecognition ele-
ment which is selective for the sequence
of DNA to be detected. It is important
that this biorecognition element is
integrated with the signal transducer.
Integration is most commonly achieved
by immobilising ss-DNA on the electrode
surface. The immobilised DNA is fre-
quently referred to as the probe strand
and the sequence to be detected is called
the target strand. To fabricate a DNA
biorecognition interface that can selec-
tively detect target DNA with a high
degree of efficiency, the immobilisation
of the probe DNA and the entire
interfacial design needs to be carefully
thought through.
First of all, the type of probe needs to
be considered. Apart from conventional
DNA there are other variants on the
nucleic acid theme that can be used as
probes, most notably PNA (peptide
nucleic acid). PNA,1114 where the
sugar-phosphate backbone is replaced
604 | Analyst, 2007, 132, 603610
with a peptide-like backbone, is of
particular interest in DNA biosensing.
The peptide backbone is neutral and
hence a PNA strand binds to DNA with
a higher affinity than DNA does itself. A
consequence of this stronger binding is
that the higher selectivity as a PNA-
modified transducer surface can
differentiate between the perfect comple-
mentary strand of target DNA and one
with a single base-pair mismatch.15
However, the majority of research has
been conducted using DNA, partly
because of the issues related to the
availability and cost of PNA, but reports
involving PNA are becoming increas-
ingly more frequent.
Once the type of probe nucleic acids
has been selected, there are number of
other important factors associated with
immobilising the DNA probe onto the
electrode surface, regardless of the type
of electrode. How the DNA is immobi-
lised, including how the probe DNA
interacts with the surface to which it is
immobilised, needs to be considered.
Much of the early work on electroche-
mical DNA biosensors, and many of the
DNA microarrays, involve immobilising
DNA to the transducer surface via
electrostatic adsorption. The merit of
such a strategy is its simplicity: no
complicated surface chemistry is required
and it is highly compatible with spotting
techniques. The drawbacks come from
the fact that for the strategy to work
DNA must have a strong affinity for the
surface. This strong affinity results in the
probe DNA being fixed onto the surface
by multiple points. Considering that
hybridisation requires the two strands
of DNA to wind around each other to
form the double helix, multipoint attach-
ment typically results in low hybridisa-
tion efficiency as the configurational
freedom of the probe strand is signifi-
cantly restricted by the immobilisation
method. In fact, multipoint attachment
of probe DNA raises the question of how
hybridisation is even possible. The nave
explanation is that as the DNA duplex is
being formed, the probe DNA partially
desorbs from the surface, winds around
the target strand to form the duplex and
re-adsorbs. Subsequently another por-
tion of the probe desorbs to allow the
hybridisation to proceed. However,
Lemeshko et al.16 have provided some
evidence on charged surfaces that in fact
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the conventional double helix does not
form but rather the target binds to the
adsorbed probe to give a highly asymme-
trical unwound duplex. The other draw-
back is the affinity of DNA for the
transducer surface meaning therefore the
target DNA will also have an affinity for
the surface, leading to non-specific
adsorption of DNA. This issue is exacer-
bated when the transduction technique
used is indicating the amount of DNA
hybridisation occurring by the amount of
DNA at a surface (see later).
The alternative to adsorption of the
DNA onto a surface is to chemically
bond the DNA to the surface, preferably
from one end only. The emphasis on end-
point attachment again relates to the
issue of hybridisation efficiency and
ensuring maximum configurational free-
dom for the probe strand. There are
several methods published in achieving
end-point attachment of DNA, which
typically relate to having a reactive group
at the end of the DNA, which is used to
covalently attach the probe DNA to an
electrode surface. The most popular
method for achieving end-point attach-
ment of probe DNA is via the synthesis
of mercaptoalkyl linkers on one end of
the DNA (thiolated-DNA). We describe
this strategy here in detail to illustrate
some of the important aspects that need
to be considered in designing DNA
recognition interfaces and want to
remind the reader that this is not the
only viable strategy for the fabrication of
DNA interfaces. This chemistry exploits
the affinity of thiols for gold, forming a
self-assembled monolayer, but can also
be used to attach DNA to maleimide-
terminated surfaces17 and epoxide-
terminated surfaces.18 The mercaptoalkyl
linker on the end of the DNA ensures a
strong bond between the DNA and the
electrode surface, but end-point attach-
ment alone is not necessarily sufficient to
ensure immobilised DNA with a high
degree of configurational freedom.
Tarlov and co-workers19,20 have ele-
gantly illustrated the impact of the
affinity of DNA for the surface on
hybridisation efficiency. Assembling
thiolated-DNA alone on the surface
resulted in a low hybridisation efficiency
with target DNA (between 0 and 5%)
which was attributed to the affinity of the
DNA bases for gold surfaces. However,
if the DNA-modified surface is incubated

in a solution of mercaptohexanol (MCH)
as a diluent layer, the affinity of the thiol
moiety on the mercaptohexanol diluent
lifts the DNA up off the gold surface and
the negative dipole of the alcohol on the
distal end of the diluent repels the
Published on 23 April 2007. Downloaded by Indian Institute of Technology Chennai on 23/10/2017 15:00:10.
negatively charged probe backbone caus-
ing the ss-DNA probe to project out in
solution. The result is an increase in the
hybridisation efficiency to almost 100%.7
A further issue to consider is the
number of base pairs the probe has.
The probe length is frequently around
1550 bases long with a linker to the thiol
of 36 carbons. This linker length can
play an important part in the sensitivity
of the sensor along with the length of
the diluent layer. Recent work by the
Gooding group21 illustrated that the
optimal arrangement is to have the linker
longer than the diluent as the further the
DNA probe is from the surface (which
really means the top of the diluent layer),
the greater the configurational freedom
of the probe and the greater the hybridi-
sation efficiency. The same conclusion is
drawn by Watterson et al.22 in that the
efficiency of hybridisation is reduced
when the target region of probe strand
is close to the surface and increased when
this target region was further away from
the surface. However, for electrochemical
biosensors there may be a cost in
increasing the length of the linker. The
longer the linker, the harder it is for
electron transfer to occur between the
electrode and the DNA. This phenom-
enon is important for several electroche-
mical DNA biosensors which use redox
reporters as is discussed below.
The surface coverage of the ss-DNA
probe, which with thiolated-DNA can be
controlled using the diluent, also has an
effect on the hybridisation efficiency of
the biosensor. It would seem that the
more probes attached to the surface the
more sensitive the biosensor will be, but
it is also necessary to maintain sufficient
space between the probes to limit repul-
sion of the incoming targets and steric
effects between the probe strands.
Peterlinz and Georgiadis23 suggest that
a surface coverage of around 5 6 1012
molecules cm22 is ideal. Repulsion of the
target also depends on the ionic strength
of the buffer solution. For a 1 M
phosphate buffer with a surface coverage
of 3 6 1012 molecules cm22 or less,
Levicky et al.24 have determined that no
This journal is The Royal Society of Chemistry 2007
significant steric or electrostatic interfer-
ence between chains would occur.
A recent strategy that uses end-point
attachment to form SAMs that could
provide exquisite control of the spacing
between probes on graphite electrodes has
recently been described.25 This method
exploits the interaction between the elec-
trode surface, highly orientated pyrolytic
graphite, and pyrenes that are attached to
the end of the probe forming self-
assembled monolayers. By altering the
size of the pyrene moiety, the change in
spacing between probes could be altered.
There are other factors that affect the
hybridisation efficiency such as salt con-
centration and temperature.8 An increase
in salt concentration gives an increase in
DNA hybrid stability26 and the rate of
hybridisation;27 however, if the detection
method (see below) uses an intercalator
such as Co(phen)33+ then lower salt
concentrations need to be considered.28
To summarise this section on the
biorecognition interface, when develop-
ing an electrochemical DNA biosensor
the researcher must consider not only
what type of probe nucleic acid to
employ and how to immobilise it but
also how the DNA will interact with the
surface, the length of the linker used to
couple the DNA to the surface, the
surface coverage of probe DNA and
the solution environment in which the
biosensor will be placed.
Approaches to achieving
transduction
Research into transducing DNA hybri-
disation has been exceedingly active;7,8
transduction strategies can be subdivided
into two main categories, label-free and
labelled approaches. Primary considera-
tions for DNA biosensors are the detec-
tion limits (as this will have a big bearing
on the extent of amplification of the
target DNA) and the selectivity. In the
ideal case a DNA biosensor would be
able to discriminate complementary
target DNA from DNA with a single
base-pair mismatch without requiring
amplification of the sample. This is a
goal that has yet to be achieved
Label-free approaches
Label-free methods for the detection of
DNA hybridisation either rely on
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changes to the electrical properties of
an interface,7 the change in flexibility
from ss-DNA to the rigid ds-DNA29,30 or
the electrochemical oxidation of guanine
bases.31,32 The latter is far and away the
most popular label-free method, partly
because of its simplicity and partly
because of its versatility, having also
been used to explore small molecules
binding with DNA.33,34 Guanine is the
most easily oxidisable base with redox
peaks being observed at around +1 V (vs.
Ag/AgCl) on most electrode materials
(more anodic peak potentials have been
observed on bamboo-like carbon nano-
tubes).35 This strategy is exemplified by
the pioneering work of Wang et al.36,37
where the probe strand has inosine bases
substituted for guanine; inosine also
binds preferentially to cytosine bases
but its oxidation signal is well separated
from the guanine peak. Thus, prior to
hybridisation with the target sequence no
guanine signal is observed. Once the
target strand is hybridised to the immo-
bilised inosine-containing probe, the
guanine peaks from the target DNA
were detected using chronopotentiome-
try. However, owing to the affinity of the
surface for DNA, non-specific binding of
the target DNA is an issue, as is the
configurational freedom of the probe
DNA. The increase in background signal
due to non-specific adsorption inevitably
increases the detection limit. Non-speci-
fic binding is a significant problem as the
technique measures the amount of gua-
nine base present on the electrode surface
regardless of whether it has hybridised
with a probe strand or has non-specifi-
cally adsorbed onto the electrode. Wang
et al. have explored strategies to resol-
ving the issue of non-specific binding by
using magnetic beads which separate and
remove the non-specifically bound DNA
from hybridised DNA using a biomag-
netic processing technology.38 Detection
limits from these types of devices are
below 100 nM,7 and when a non-
complementary strand of DNA was
introduced a substantially smaller gua-
nine signal was observed than for a
complementary strand.
Label-free methods can be extended to
using electrochemical impedance spectro-
scopy (EIS).7,3950 This method measures
the change in the faradaic impedance, in
the presence of a redox species such as
ferricyanide, from an ss-DNA-modified
Analyst, 2007, 132, 603610 | 605

electrode to its hybridised version. Owing
to the repulsion of ferricyanide by the
negatively charged duplex, an increase in
charge transfer resistance is observed.
Work in this area first concentrated on
the amplification of the signal by exploit-
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ing the extra length of the target strand
beyond the probe.40 This extra length can
be hybridised to a third strand with
biotin attached to one end. Avidin can
then bind to this strand, causing a greater
increase in the charge transfer resistance.
The attachment of further biotins via
liposomes gives even greater amplifica-
tion.49 More recently, the use of EIS has
been developed with PNA/DNA biosen-
sors,46 conducting polymers47 and gold
nanoparticle-based sensors.45 The latter
method involves attachment of gold
nanoparticles to a bilayer of a thiol-
containing silane immobilised on a gold
electrode. The ss-DNA can then be
attached to the nanoparticle without
losing its ability to bind the target strand,
giving a detection limit of 5 nM.
Labelled approaches
Labelled methods are significantly more
popular than label-free approaches
because there are many more ways in
which transduction can be configured
with high sensitivity and selectivity. In
electrochemical DNA biosensors the
labels are typically redox active mole-
cules36,5153 which either intercalate
between the WatsonCrick base pairs of
a DNA duplex, and hence do not interact
significantly with ss-DNA, or they are
molecules that bind preferentially to
either ss-DNA or ds-DNA. There have
also been a number of reported strategies
where the labels are enzymes,54,55 nano-
particles38,5661 or liposomes.49,50
The first electrochemical DNA hybri-
disation biosensor by Mikkelsen and co-
workers,36 where Co(Phen)33+ and
Co(bpy)33+ bound to the minor groove
of ds-DNA is an example of where the
label has a higher affinity for ds-DNA
over ss-DNA. Recent developments in
this area include the synthesis of a new
cobalt groove binder62 for the detection
of sequences from the HIV DNA show-
ing a detection limit of 27 pM.
The work of Ozsoz and co-workers
using the redox marker Methylene Blue
in the detection of sequences related to
the hepatitis B virus51 is one of the best
606 | Analyst, 2007, 132, 603610
developed examples of the preferential
binding strategy. The important differ-
ence from the Mikkelsen approach is
that the redox marker has a higher
affinity for the ss-DNA as distinct from
the ds-DNA. Methylene Blue (MB) can
act as a DNA intercalator, but in this
work it shows an affinity for exposed
guanine bases.63 Therefore, upon
hybridisation, there is a decrease in the
MB signal as the access of MB to the
guanine bases is restricted. There is a
linear relationship between the number
of guanine bases in the immobilised
probe and the amount of charge passed
which allows the determination of the
number of probes immobilised if the
number of guanines per probe is
known.63
All the methods of detecting DNA
hybridisation discussed thus far indicate
hybridisation due to a difference in
electrochemical signal based on a change
in the amount of DNA at an electrode
surface. More recently, electrochemical
biosensors have been developed where
the electrochemical signal is actually
dependent on the formation of a DNA
duplex. This can be achieved either by
using electrochemistry to monitor the
change in flexibility of DNA upon
hybridisation or by probing the change
in electron transfer properties.
Physical changes in DNA from the
flexible ss-DNA to the rigid rod-like
ds-DNA can be used for detection in
labelled methods, as well as the label-free
methods mentioned above, by attaching
a ferrocene tag to the end of the
immobilised probe.30,64 The flexibility of
ss-DNA enables the ferrocene tag to
come within close proximity of the
electrode surface such that it can be
oxidised and reduced. Upon hybridisa-
tion, however, the rigid DNA duplex
stands normal to the electrode surface.
This increases the electron transfer dis-
tance from the electrode to the ferrocene
tag to well beyond where any appreciable
electron transfer can be observed. Heeger
and co-workers65,66 improved these ideas
to ensure that the ferrocene was near the
electrode surface before hybridisation. A
ferrocene tag is attached to a DNA stem-
loop structure immobilised on gold
(Fig. 2); before hybridisation, the loop
is arranged such that a section of it
hybridises to itself, bringing the
ferrocene tag into close contact with the
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gold electrode and giving a good
electrochemical signal. Once hybridisa-
tion occurs, the DNA again projects
normal to the surface, distancing the
ferrocene from the electrode surface and
hence switching off the electrochemical
signal. Although this method gives a
signal off device, which is prone to false
positives, its detection limit of 10 pM 66 is
encouraging. Recently, the Heeger group
has further improved the idea to give a
signal on device by using target-induced
strand displacement.67
A particularly powerful strategy to
transduce DNA hybridisation is by the
change in charge transfer properties
between a single strand and a double
strand of DNA. Long-range charge
transfer through DNA can be used not
only to transduce that a DNA duplex has
formed but also to provide information
on the quality of the duplex formed. The
result is a DNA biosensor with superior
selectivity and, in some cases, simplicity
of operation. This strategy was pioneered
by Barton and co-workers6870 using
intercalators such as Methylene Blue or
Daunomycin. The strategy is reliant on
the ability of ds-DNA to act as a conduit
for charge transfer over distances of 40 A
or more,71 an ability which ss-DNA does
not possess. Thus, when a DNA duplex is
formed, the MB or daunomycin inter-
calate into the DNA duplex and can be
oxidised via charge transfer through the
DNA. If the probe DNA has not
hybridised with the target then a greatly
reduced current was observed. Single
base-pair mismatches were detected using
this strategy without the need for any of
the stringency washings, (stringency
washings are required to differentiate
between perfectly complementary strands
and mismatch targets with most other
systems). The absence of the need for
stringency washings greatly simplifies the
operation of this electrochemical DNA
biosensor.
Related work by Wong and Gooding72
has improved both the robustness and the
simplicity of the charge transfer approach
using the negatively charged inter-
calators anthraquinonemonosulfonic acid
(AQMS; anthraquinone-2-sulfonic acid)73
or anthraquinone-2,6-disulfonic acid
(ADQS).21,72 Fig. 3 depicts how the
DNA biosensor is operated. A DNA
recognition interface is prepared using
the method of Tarlov and co-workers,19
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nanoparticles.38,5661 This method is

based on the hybridisation of biotiny-
lated target DNA to detect DNA immo-
bilised onto a magnetic bead.
Streptavidin-modified gold nanoparticles
were then added, which bound the
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biotinylated target DNA. Hybridisation

is then detected by dissolving the gold
nanoparticles with acid and carrying out
stripping potentiometric analysis at car-
bon electrodes56 (see Fig. 4). Other
similar methods of carrying out the
detection have also been developed.58,60
A label-free method of detecting hybri-
disation has also been developed based
on the inosine methods mentioned ear-
lier. First, the non-hybridised targets are
magnetically removed before denaturing
the hybrid and detecting guanine oxida-
tion56 which is amplified by the addition
of copper.61 These multi-step techniques
give detection limits with the copper
amplification of 0.8 nM.

Fig. 2 (A) Schematic of the DNA stem loop strategy for detecting DNA hybridisation by Future directions
Heeger and co-workers.65,66 A ferrocene tag attached to the end of the DNA stem loop is
Enormous progress has been made in the
electrochemically accessible. Upon hybridisation the ferrocene is removed from the
electrode surface and the electrochemical signal is turned off. (B) Voltammograms development of electrochemical DNA
(background-subtracted) of the sensor containing a range of complementary DNA biosensors but there are still many
concentrations; from top to bottom: 0 M, 30 pM, 500 pM, 30 nM, 800 nM, 5 mM. Inset hurdles to be overcome for portable
shows a calibration curve of peak current with complementary DNA concentration. (Fig. 2 electrochemical DNA biosensors to
reproduced with permission from ref. 66. Copyright 2003, National Academy of Sciences, become widely commercialised. The first
USA.) challenge relates to the fact that DNA
biosensors require some sample prepara-
tion because, unlike many other types of
where thiolated-DNA is assembled onto be differentiated from the current due biosensors, the sample to be analysed is
a gold electrode surface followed by to AQMS in solution, accessing the itself not readily available. All the
MCH as the diluent. The sensor is electrode directly. The final DNA bio- systems discussed above only focus on
exposed to a sample of DNA and to sensor gives a response within one the analysis of a prepared sample; how-
the AQMS intercalator simultaneously hour, can differentiate between comple- ever, much work has been done on
and after a defined time period (one mentary sequences of DNA and both developing miniaturised methods of sam-
hour for the most favourable interface CA and GA mismatches and has a ple preparation and amplification.74,75
for DNA hybridisation) long-range detection limit of 0.5 nM. Furthermore, Even then, the majority of biosensors
charge transfer through ds-DNA allows this DNA biosensor allows DNA hybri- developed thus far possess either good
hybridisation to be transduced. This is disation to be monitored in real time.21 sensitivity or selectivity, and so, in the
possibly the simplest DNA biosensor to The long-range charge transfer strat- future, work needs to be directed towards
operate yet developed. The simplicity egy is one way of improving the selectiv- combining these issues. Sensitivity is
arises from the target DNA and the ity and simplicity of electrochemical an issue since amplification using the
label all being present in the same DNA hybridisation biosensors but per- polymerase chain reaction (PCR) is
solution so there is only one step to haps at the cost of sensitivity. Other usually required to increase copy num-
operate, exposing the biosensor to the methods have made a conscious decision bers, but the amplification process can
sample. This simplicity arises from an not to aim for a one-step procedure, also cause amplification of contami-
anodic shift in the oxidation potential considering any DNA assay will require nants. Reducing the detection limit
of AQMS upon intercalation compared sample preparation to get the DNA out decreases the number of amplification
with when in bulk solution (Fig. 3). of the cell and ready for detection, and cycles and hence alleviates this problem.
This potential shift, attributed to the hence concentrate on sensitivity and Work is required on detecting target
change in environment of the AQMS selectivity instead. A nice example of DNA from a mixture of multiple
upon intercalation, means that the this approach is by Wang et al. who sequences and is something that needs
current due to intercalated AQMS can developed multi-step arrays using to be developed. In some respects this

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Developing this further, certain proteins,

such as MutY, contain a redox centre
that is capable of binding to the DNA
duplex allowing electron transfer to the
electrode and an electrochemical signal
to be detected.79 Other work on protein
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detection by Wangs group80 uses apta-

mers. Aptamers are sequences of nucleic
acids that selectively bind to proteins. A
biotinylated aptamer is attached to mag-
netic beads that have been coated with
streptavidin. The attached aptamers then
bind the target protein. This is followed
by magnetic separation of the beads to
which the aptamer and protein are
attached. The protein is then released
using NaOH and is detected via a
chronopotentiometric stripping method.
This method of protein detection is
related to other work by Wang et al. on
nanoparticles, discussed above, and gives
detection limits of around 7 nM. Kraatz
et al. have also worked in this field and
used the protein MutS to determine
single base mismatch as MutS only binds
to duplexes containing a mismatch.81 For
all these detection methods, surface
Fig. 3 (A) Electrochemical DNA biosensor based on long-range charge transfer by Wong control is important to allow room for
et al.73 where the hybridisation of the target, intercalation of the redox molecule AQMS and proteins to bind.
the electrochemical measurement were carried out simultaneously. (B) Osteryoung square
Anti-cancer drugs, such as cis-platin,
wave voltammetry results showing the appearance of the shoulder peak due to the
target genetic diseases by binding to
intercalation of AQMS into the DNA duplex. Inset shows background-subtracted shoulder
peak. (Fig. 3B reprinted with permission from ref. 73. Copyright 2006, American Chemical DNA through intercalation or binding
Society.) to the DNA grooves, and information on
the efficiency of these drugs and
improvements in the drug design can be
work has already started.21,76 For proteinDNA interactions have in fun- investigated using electrochemical DNA
example, a long-range charge transfer damental cellular processes, such as biosensors.82 The binding of the small
method using AQMS has already been transcription. Studies of DNAprotein molecules to DNA causes a decrease in
shown to be able to selectively detect interactions are commonly carried out by the guanine signal when the drug binds
target DNA without the loss of signal, techniques such as gel retardation.78,90 to ds-DNA.83 These biosensors have also
even in a solution containing a mixture However, Barton and co-workers77,78 been used in drug activity studies, such as
of excess salmon testes, salmon sperm, have employed their long-range charge the work carried out by Ozsoz and co-
calf thymus or non-complementary transfer biosensor to explore the interac- workers84 in the development of novel
oligonucleotides.76 tions of proteins with ds-DNA: the redox anti-cancer drugs related to cis-platin
What is the future of electrochemical label, daunomycin, is covalently linked such as cis-bis(3-aminoflavone)dichloro-
DNA biosensors? Recently, work in this to the DNA at the top of the film and platinum(II) which exploits the decrease
field appears to be moving beyond immobilised onto a gold surface with a in adenine oxidation signals on the
hybridisation. There are analytes other diluent such as MCH. In one example, binding of these drugs.
than DNA that these biosensors can be the binding of the protein methyltrans- DNA damage and interaction of drugs
employed to detect. A few examples are ferase Hhal to a specific sequence of ds- such as the anti-tumour antibiotic mitox-
proteins,7781 which can be at abnormal DNA is monitored by the decrease in the antrone with DNA duplexes has been
levels in the presence of cancer, charge transfer through the DNA, and investigated also by Oliveira-Brett
drugs such as the anti-cancer drug hence the electrochemical signal, due to et al.9193 This work explores the
cis-platin,8284 and phenotyping, which the protein catalysing the methylation of decrease of guanine and adenine signals
still detects DNA damage but not via cytosine bases. The methylation of cyto- on the binding of these drugs to the bases
hybridisation.79,8589 sine hence disrupts the base-pair stack- to investigate possible DNA damage that
Investigating the interaction of DNA ing. With a protein that does not bind to might be caused by such drugs.
binding proteins with DNA is important ds-DNA such as bovine serum albumin The other trend that seems to be
due to the significant role that these (BSA), no such current drop is observed. emerging is the enzymatic modification

608 | Analyst, 2007, 132, 603610 This journal is The Royal Society of Chemistry 2007

Published on 23 April 2007. Downloaded by Indian Institute of Technology Chennai on 23/10/2017 15:00:10.
Fig. 4 Steps involved in nanoparticle-based detection developed by Wang et al.:56 (a)
streptavidin-coated bead; (b) biotinylated probe immobilised onto the magnetic bead; (c) the
biotinylated target is hybridised to the probe; (d) capture of streptavidin-coated gold
nanoparticles by the hybridised target; (e) the gold tag is dissolved and detection by
potentiometric stripping analysis occurs. (Fig. 4 reprinted with permission from ref. 56.
Copyright 2001, American Chemical Society.)
of DNA with labelled bases for pheno-
typing of SNPs by the extension of a
DNA template. Generally this requires
enhancing the redox properties of DNA
by developing electroactive nucleotides
and incorporating these into DNA
strands. King and co-workers79,85,86 have
developed ferrocene- and anthraquinone-
labelled nucleotides that have been
incorporated in DNA and RNA. These
labelled nucleotides were incorporated
using single base extension for the detec-
tion of SNPs: the different ferrocene and
anthraquinone labels gave distinguish-
able electrochemical signals, which
would allow different bases to be
detected. Work carried out by Brazill
et al.87,88 uses single base extension with
capillary gel electrophoresis and end
column electrochemical detection, again
for SNP detection. Patolsky et al.89
extend viral DNA hybridised to a probe
immobilised on a gold electrode surface
with dNTPs and ferrocene-labelled
dUTP then use GOx to catalyse trans-
formation of ferrocene to give an ampli-
fied signal. This allows the detection of
the viral DNA as the extension can only
occur with the template (the viral DNA)
hybridised to the probe.
This journal is The Royal Society of Chemistry 2007
The message from this review we hope
the reader has absorbed is that tremen-
dous progress has been made into devel-
oping electrochemical DNA biosensors,
but equally well huge opportunities exist
for new workers in the field. The field is
sufficiently mature that many concepts
have been developed and many of the
hurdles that must be overcome have been
identified. So we approach an exciting
time where solutions to these hurdles are
being found and commercial devices may
be part of the near future.
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