Talanta 56 (2002) 809– 819

www.elsevier.com/locate/talanta

Review paper

Past, present and future of nucleic acids electrochemistry

Emil Paleček *
Institute of Biophysics, Academy of Sciences of the Czech Republic, Králo6opolská 135, CZ-612 65 Brno, Czech Republic
Received 26 October 2001; accepted 15 November 2001

Abstract

Electrochemistry of nucleic acids was discovered about 40 years ago. During the first 15 years electrochemistry
brought early evidence of DNA premelting and polymorphy of the DNA double helix. At present electrochemical
methods working with stationary electrodes are able to detect DNA at attomol and in some cases, even at lower
levels. A great progress in the development of electrochemical sensors for DNA hybridization and DNA damage
achieved in recent years suggests that these sensors may soon become important tools in medicine and other areas of
practical life of the 21st century. © 2002 Elsevier Science B.V. All rights reserved.

Keywords: Nucleic acid electrochemistry; History; DNA sensors; DNA hybridization and damage

1. Introduction believed that simple devices for determining NA

At present electrochemistry of nucleic acids
(NAs) is booming due to the expectations of the
development of electrochemical transducer-based
devices for detection of nucleotide sequences and
DNA damage [1–3]. Better diagnosing, prevent-
ing and treating many human diseases will require
efficient and inexpensive tools for determining
DNA and RNA sequences. Such tools will find
use also in other applications such as veterinary
and forensic medicine, environmental testing etc.
To keep our environment clean, rapid and accu-
rate assays for DNA damage are needed. The
main advantages of electrochemical devices are
their low-cost, fast response, simple design, small
dimensions and low power requirements. It is
* Tel.: +420-5-492-46241; fax: +420-5-412-11293.
E-mail address: palecek@ibp.cz (E. Paleček).
nucleotide sequences will reach the doctor’s offices
within few decades.
2. History
2.1. Electroacti6ity of nucleic acids
As a graduate student I found in 1957 that
DNA and RNA yielded reduction and oxidation
signals upon interaction with electrodes [4,5]. It
was the so-called oscillographic polarography at
controlled a.c., introduced by J. Heyrovsky [6,7]
in the beginning of the 1940s (constant a.c.
chronopotentiometry according to the present
nomenclature) [8], that proved to be better suited
for the analysis of NAs than the d.c. polarogra-
phy, popular at that time. I can hardly forget the
interest of Professor Jaroslav Heyrovsky and sup-

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PII: S 0 0 3 9 - 9 1 4 0 ( 0 1 ) 0 0 6 4 9 - X
810 E. Paleček / Talanta 56 (2002) 809–819
port I obtained from him, his assistant Dr Robert
Kalvoda and his son Dr Michael Heyrovsky.
To my knowledge, the first paper dealing with
electrochemical analysis of nucleic acids (NAs)
was published by Berg [9]. He used supporting
electrolyte containing cobalt ions to determine
proteins in RNA and DNA samples and under
the given conditions he found the NAs as polaro-
graphically inactive species. His finding was in a
good agreement with the literature claiming that
among the nucleic acid components adenine is
polarographically reducible only at highly acidic
pH values [10] while the other components are
inactive. In collaboration with my colleague B.
Janik we showed that in addition to adenine (A)
also cytosine (C) was reduced at the dropping
mercury electrode (DME); reduction of the latter
base took place not only at acid but also at
neutral pH [4,5,11–13]. Guanine (G) produced a
specific anodic signal, [4,11] explained by oxida-
tion of the DNA reduction product formed at
highly negative potentials [4,5,11,14]. B. Janı́k
later joined P. Elving’s laboratory which signifi-
cantly contributed to the present knowledge of
electrochemistry of the NA components (reviewed
in Ref. [15]). Moreover, all NA bases and some
other purine and pyrimidine derivatives produced
anodic signals due to the formation of sparingly
soluble compounds with the electrode mercury
which were later utilized for the cathodic stripping
voltammetric determination of NA bases at
nanomolar concentrations [16– 18]. The ability of
NA bases to associate at the electrode surface was
demonstrated by V. Vetterl in the second half of
the 1960s [19–21].
2.2. Electrochemical signals depend on the DNA
structure in solution
I found that signals of A, C and G residues can
be observed at oscillopolarograms dE/di = f(E) of
single-stranded (ss, denatured) DNA while with
the double-stranded (ds, native) DNA these sig-
nals were smaller or absent (Fig. 1). Soon it
became clear that the electrochemical signals ob-
tained with mercury electrodes were very sensitive
to small changes in the DNA structure in solu-
tion. Shortly after publishing my paper on oscillo-
graphic polarography of calf thymus DNA and its
degradation products in Nature (1960) [5] I was
invited by Julius Marmur from the Harvard Uni-
versity to work in his laboratory as a postdoctoral
fellow. It took about 2 years before I was allowed
to leave the Party/Government-ruled Czechoslo-
vakia but to me it was like a miracle. At that time
it was possible to follow DNA denaturation and
renaturation with oscillopolarography. That tech-
nique showed also a new phenomenon— the
DNA premelting. Knowing the work of my future
boss who discovered, together with Paul Doty, the
DNA renaturation [22] and proposed a method of
DNA isolation from bacteria [23] (in that time his
Fig. 1. Scheme of a controlled a.c. oscillopolarogram dE/dt
against E of double stranded (ds) and single stranded (ss)
DNAs using single-sweep (first curve) technique at neutral pH
(reviewed in [115]). Cathodic capacitive CI-1 and faradaic CI-2
indentations as well as anodic indentation AI are shown. CI-2
is characteristic for ss (denatured) DNA and corresponds to
CV peak CA [3,5,25,39]; CI-2 and (peak CA on cyclic voltam-
mograms [3,116]) is due to the reduction of adenine and
cytosine residues and it strongly decreases or disappears (----)
in the second cycle (second curve) due to blocking of the
electrode surface by the DNA reduction product [25,116]. CI-2
is absent at in ds (native) DNA. AI is produced both by ss and
dsDNAs and corresponds to CV peak G which are due to the
oxidation of the guanine reduction product formed at highly
negative potentials around −1.8 V (at neutral pH, against
SCE); AI and peak are G substantially smaller in dsDNA than
in ssDNA. At alkaline pH’s all free nucleic acid bases pro-
duced indentations [4,11] at positive potentials (----) due to
formation of sparingly soluble compounds with the electrode
mercury. These signals were utilized in the cathodic stripping
voltammetry determination of bases at ppb level [16 – 18,117 –
120] and for detection of the DNA hybridization [73].
 E. Paleček / Talanta 56 (2002) 809–819
laboratory was perhaps the world largest stock of
pure, native bacterial DNA samples with different
base contents), I was excited by the opportunity
that lied ahead of me. In 1962 the instrument for
the oscillographic polarography ‘Polaroskop P
524’, was produced only in Czechoslovakia. J.
Marmur, who in the meantime moved from Har-
vard to Brandeis University, advised me to bring
the instrument with me. So, I sent it as an air-
cargo. Before my departure we constructed in my
laboratory a new multi functional instrument us-
ing rectangular a.c. capable of displaying single-
sweep oscillograms at different frequencies, as
well as different functions in addition to dE/dt
against E, etc. I was, however, not allowed to take
the instrument out of the country. Equipped with
letter of recommendation from J. Heyrovsky I
went to U.S. in November 1962 with no doubts
about the suitability of my electrochemical
method for the nucleic acid research. Unfortu-
nately, it took about 9 months for the instrument
to arrive, completely broken. In the laboratory
the opinion prevailed that the instrument was
thoroughly ‘searched’ by secret service on both
sides of the ‘Iron Curtain’.
In the meantime, I learned some microbiology
and took part in a purely molecular biological
studies concerned with RNA transcription [24].
At the end of my stay I managed to fix the
instrument and to do some DNA measurements
which I finished at home. Those results showed
that compared to DNA melting, the DNA
premelting was strongly dependent on the nucle-
otide sequence of bacterial DNAs [25].1 Great
opportunity for the electrochemical analysis of
DNA was thus almost lost in 1963. I continued
my work at home under the usual conditions of
the lack of chemicals and scientific information
from the West.
Derivative pulse polarography (DPP) was ap-
plied for the first time in the analysis of DNA in
1965. Great difference in the DPP peak heights of
ss and dsDNAs was utilized in a highly sensitive
determination of traces of ssDNA in the presence
1
Oscillopolarographic experiments on interaction of DNA
with methylene blue, done in collaboration with Melvin Simon
(now Professor at Caltech), have never been published.
811
of a large excess of dsDNA [26]. In the 1960s and
in the first half of the 1970s electrochemical tech-
niques, and particularly DPP, produced early evi-
dence not only of DNA premelting but also of
polymorphy of the DNA double helix (reviewed
in [27]. The polymorphy of the DNA structure
was later well established by the X-ray crystallog-
raphy and NMR [28,29]. Further studies of DNA
premelting are, however, needed and it may be
expected that the present electrochemical tech-
niques may help to shed more light on the previ-
ous results and to contribute to a better
understanding of the DNA properties in solution
and at charged surfaces.
In 1975, I organized a symposium on ‘Electro-
chemistry of Nucleic Acids’ which was attended
by almost all scientists interested in nucleic acid
electrochemistry at that time, including G.C.
Barker, H. Berg, G. Dryhurst, J. Flemming and
H. W. Nürnberg. My graduate student V. Brabec
took part also at this meeting, becoming later a
postdoc of G. Dryhurst with whom he introduced
carbon electrodes in the research of polynucle-
otides [30], few years after the first application of
the hanging mercury drop electrode (HMDE) by
Nürnberg et al. [31–34] and by Berg and Flem-
ming [35] to the NA research. The results of
conventional electrochemical analysis of NAs
with the mercury or carbon electrodes immersed
in the NA solution during the electrochemical
measurements were thoroughly reviewed [32,35–
39].
3. Effect of the electrode potential on the DNA
structure
A good correlation between polarographic re-
sponses (using small potential excursions during
the life time of the dropping mercury electrode)
on one hand and optical methods on the other
hand suggested that the structure of dsDNA is
not significantly changed due to adsorption of
dsDNA at DME around − 1.4 V where the DNA
reduction signals were measured. It was found,
however, that in a narrow potential range, close
to the potential of dsDNA desorption (around
− 1.2 V), large changes in the interfacial proper-
812 E. Paleček / Talanta 56 (2002) 809–819
ties of dsDNA occurred involving major parts of
the adsorbed DNA molecules, if dsDNA was
exposed to these potentials for sufficiently long
time (about 1– 2 min) (reviewed in Ref. [36]). In
1974 these changes were interpreted in terms of
opening of the DNA double helix at the nega-
tively charged electrode surface, due to the effect
of the repulsive potential on dsDNA anchored at
the mercury surface via hydrophobic bases re-
leased from the duplex, e.g. at the strand interrup-
tions [40] (reviewed in Ref. [36]). At acid pH’s the
DNA surface denaturation occurred in a wider
potential range [41]. Other explanations were of-
fered in that time by Berg’s group [35] considering
some hypothetical properties of DNA at the sur-
face including conductivity of randomly oriented
chromosomal DNA (reviewed in [35,36].
Hartwich et al. [42] have recently shown that such
DNA adsorbed at the electrode surface is, how-
ever, an insulator. Recent experiments with DNA-
modified electrodes [43] and particularly with
covalently closed circular DNAs strongly support
the opening [44–49] of the DNA double helix at
the electrode surface. Potential-controlled opening
of dsDNA appears very attractive in connection
with the recent attempts to create DNA biotech-
nologies on chips involving fast DNA sequencing;
more research in this interesting area can thus be
expected in the near future.2
4. Adsorptive transfer voltammetry and
NA-modified electrodes
At the beginning of the 1980s, the importance
of electrochemical techniques for the DNA bio-
chemical and molecular biological studies de-
creased rapidly as the DNA research became
oriented to the studies of well-defined oligonucle-
otides and plasmid or viral DNAs of known
2
After finishing this article, I noticed a recent paper by
Heaton et al. [50] showing opening of the ds DNA at a
negatively charged gold surface induced by the electric field.
The mechanisms responsible for the DNA opening at mercury
and gold electrodes can hardly be exactly the same but in both
cases the effect of the electrostatic field on the DNA duplex
anchored at negatively charged electrodes is involved.
nucleotide sequences. The preparation of such
DNA samples was laborious and expensive and
therefore, more sensitive biochemical methods
were preferred. In the middle of the 1980s we
introduced in the nucleic acid (NA) voltammetric
analysis the adsorptive stripping techniques, in-
creasing the sensitivity of the DNA determination
by several orders of magnitude. At the same time
we started our work with the DNA-modified elec-
trodes [51–55] and within less than 10 years the
DNA-modified electrodes dominated the field,
forming solid base for the development of the
DNA detectors and sensors (reviewed in [3,39]). It
was found that DNA and RNA can be easily
immobilized at mercury and carbon electrodes
simply by immersing the electrode in a small drop
(3–10 ml) of a NA solution for a short time
(usually 30– 180 s) [44,51,53,54]. Due to strong
adsorption of DNA or RNA a stable layer was
formed at the electrode surface; the electrode was
then washed and the voltammetric measurements
were performed in solutions not containing any
NA. In this arrangement, the volume of the ana-
lyte was reduced by 2–3 orders of magnitude as
compared to the conventional voltammetry. The
former technique has been called Adsorptive
Transfer Stripping Voltammetry (AdTSV). Com-
bination of AdTS with the constant current
chronopotentiometry has been particularly suc-
cessful in the analysis of nucleic acids [56–58] and
proteins [59–61] at carbon and mercury elec-
trodes. Later covalent immobilization of NAs at
electrodes (e.g. binding of sulfur end-labeled
oligonucleotides at gold electrodes) has become
popular (reviewed in Ref. [1– 3]).
5. Electroactive markers of nucleic acids
Nucleic acids are electroactive but their reduc-
tion and oxidation at electrodes is irreversible
producing signals at highly negative or positive
potentials (reviewed in [3,36,39]). Attempts have
been therefore, made to use complexation and
derivatization of NAs to obtain reversible pro-
cesses at less extreme potentials or catalytic sig-
nals providing higher sensitivity and/or better
selectivity for the NA structure. It was Berg who
 E. Paleček / Talanta 56 (2002) 809–819
by the end of the 1960s pioneered the electro-
chemical studies of DNA interactions with elec-
troactive intercalative drugs and other
compounds binding noncovalently to DNA
[62,63]. Later, studies of the DNA interactions
with small molecules, such as intercalators and
grove binders intensified in connection with
search for redox indicators for DNA hybridiza-
tion sensors (reviewed in [2,3]).
5.1. Co6alently bound markers
The first electroactive markers covalently
bound to DNA were used at the beginning of
the 1980s [64–67]. They were based on osmium
tetroxide complexes with nitrogen ligands (Os,L)
forming stable DNA-Os,L adducts which pro-
duced redox couples and yielded also a catalytic
signal at about − 1.2 V at the mercury electrode;
moreover, these complexes showed a useful selec-
tivity for ssDNA. It was the first electrochemical
studies of the osmium tetroxide, 2,2%-bipyridine
(Os,bipy) and other Os,L complexes which led
the way to a wide application of these com-
pounds as probes of the DNA structure in vitro
and in vivo in connection with nonelectrochemi-
cal methods including DNA sequencing tech-
niques and immunoassays (reviewed in Refs.
[28,68,69]). Most of the DNA-Os,L adducts, such
as Os,2,2%-bipyridine (Os, bipy), bind preferen-
tially to thymines in ssDNA and can be deter-
mined electrochemically at, or in some cases even
below ppb level [70,71]. In AdTSV experiments
with 3 ml analyte volume, hundreds of attomoles
of ss 20-mer oligodeoxynucleotides (ODN) are
sufficient for analysis at short waiting times (de-
pending on the ODN base composition). In the
1990s ferrocenyl derivatives, daunomycin and
other compounds producing reversible couples
were covalently bound to the ends of ODNs and
used in DNA hybridization studies (reviewed in
Ref. [2]).
6. DNA sensors
At present time the DNA sensor research is
focused on sensors and detectors for DNA hy-
bridization and for DNA damage.
813
6.1. Sensors for DNA hybridization
The ability of electrochemical analysis to trace
DNA renaturation was found [27,72] shortly af-
ter the discovery of the ability of ssDNA to
reform (renature) its double-helical structure by
Marmur and Doty (reviewed in Ref. [22]). The
principle of DNA renaturation and hybridization
was exploited in many molecular–biological
methods, including DNA amplification by PCR,
DNA sequencing and in sensors for DNA hy-
bridization. In these sensors ssDNA is used as a
recognition layer (DNA probe) capable to iden-
tify a nucleotide sequence of target DNA which
is complementary to the DNA probe. In an elec-
trochemical biosensor, a DNA probe (which is a
short ss ODN) is immobilized on an electrode
(transducer) to create the recognition layer (see
Figure 1 in Ref. [73] in this issue). The DNA-
modified electrode is then dipped into a solution
of target DNA to test its nucleotide sequence.
When the sequence of target DNA exactly
matches that of the probe (based on the comple-
mentarity principle stating that G pairs with C
and A with T), a hybrid (probe-target) duplex
DNA forms at the electrode surface. Two most
important steps in the detection of the DNA
nucleotide sequence are taking place: (a) forma-
tion of the hybrid duplex DNA at the recogni-
tion layer; and (b) transformation of the latter
event into an electrical signal. The early papers
on the DNA hybridization at electrodes were
reviewed by Mikkelsen [74]. Recent progress in
the development of the electrochemical DNA hy-
bridization sensors was summarized in several
reviews [1–3]. One of the most important break-
through was perhaps the first detection of the
single base mismatch (DNA point mutation) by
means of an electrochemical detector using the
peptide nucleic acid (PNA) as a probe [75,76].
Electrochemical behavior of PNA has been then
studied in a greater detail [77–79]. At present the
DNA single base mismatches can be distin-
guished from the fully matched duplex by their
different thermostabilities without using PNA
[73,80]. Almost all papers oriented towards the
814 E. Paleček / Talanta 56 (2002) 809–819
development of the DNA hybridization sensors
dealt with model, relatively short synthetic ODNs
used as probes interacting with target DNAs of
about the same lengths. Now the studies should
focus on the analysis of natural target DNAs both
directly isolated from organisms and organs, in-
cluding human blood, as well as on shorter pieces
of DNA and PCR products.
6.2. Sensors for DNA damage
DNA damage causes serious disturbances in
basic living processes. Damage to DNA bases can
lead to alterations in base pairing and to muta-
tions. Formation of DNA strand breaks may
result in chromosome aberrations. Accumulation
of mutations and/or other kinds of DNA damage
increases carcinogenic or teratogenic risks. Elec-
trochemical methods can be used: (a) for the
detection of DNA strand breaks and base dam-
age; and (b) for detection of electroactive sub-
stances that specifically interact with DNA
(covalently and/or noncovalently). The usefulness
of the electrochemical detectors or sensors for
DNA damage is closely connected with their sen-
sitivity; a number of approaches and methods for
the detection of the DNA strand breaks and base
damage have been proposed but their sensitivity
was rather low. Recently Zhou and Rusling [81]
showed that catalytic square wave voltammetric
oxidation using Ru(bipy)23 + provided a promising
tool for detecting chemically induced DNA dam-
age. Using this technique detection of 0.1% of
damaged bases was possible (providing about
three-fold improvement over direct oxidation by
square wave voltammetry (SWV) [82]). Even this
improved sensitivity does not, however, corre-
spond to the present requirements, which have
been met only by the detectors for DNA strand
breaks using a DNA-modified mercury electrodes
(one DNA strand break among \ 2 × 105 intact
sugarphosphate bonds) [45– 49,53,83]. The
reader can find more details in the recent review
[3]. The area of the electrochemical detection of
the DNA damage is very promising and impor-
tant breakthroughs can be expected in the coming
years.
7. Commercialization
To my knowledge among the most important
companies involved in the development of the
DNA hybridization sensors are Clinical Microsen-
sors/Motorola, november AG (cooperating with
Siemens AG Medical Solution) and Xanthon.
Two of them sent a contribution to this issue.
While it cannot be expected that they would
disclose the proprietary information, they send a
clear message that their research is oriented to the
development of an electrochemical DNA hy-
bridization device that can be commercialized.
One of the most important parts of such a device
will be the working electrode (transducer). From
the available information one can guess that dis-
posable gold, carbon (plastic or screen-printed) or
indium tin oxide (ITO) are good candidates for a
transducer in such a device. Our results [73,84]
suggest that mercury and mercury amalgam elec-
trodes may soon join this triumvirate. On the
other hand, new researchers and companies are
entering the area and it is possible that their ideas
and efforts will dramatically change the present
situation.
8. Present state reflected in this issue
Contributions in this issue are closely connected
to the problems of the DNA sensors development.
Some of them are concerned with improvements
of the detection of the DNA damage [84– 86]
other consider either the questions of the elec-
trode materials and procedures suitable for the
DNA analysis [87–93] or show progress achieved
in the development of the DNA hybridization
sensors [73,94–99]. Particularly interesting are the
papers by Willner et al. [95], Ihara et al. [97], and
Popovich et al. [96] These authors are showing the
recent progress in perhaps the most perspective
elctrochemical technologies whose principles were
previously published (reviewed in [2,3]).
Willner et al. [95] describe three different meth-
ods for the detection of a single-base mismatch in
DNA by means of quartz–crystal-microbalance.
One of these methods is extremely sensitive allow-
ing detection of the single-base mismatch in the
 E. Paleček / Talanta 56 (2002) 809–819
analyzed DNA at a concentration of 3× 10 − 16 M
(in a 0.5 ml volume). This method is based on
association of a Au-nanoparticle/avidin conjugate
to the biotinylated base at the mismatched site
and on catalyzed deposition of gold on the
nanoparticle. The experiments were done only
with relatively short target ODNs (41 bases) but
such a high sensitivity gives hope for future nucle-
otide sequence detection without DNA
amplification.
Popovich et al. [96] summarize the state of art
of technology based on catalytic oxidation of
guanine at ITO originally developed by Thorp et
al. (reviewed in [1]), and show advantages of
binding the probe DNA to ITO through a self-as-
sembled phosphonate monolayer. Thorp’s label-
free technology requires absence of guanine in
probe DNAs to obtain the best resolution. This is
a disadvantage when working with short target
DNAs but if long target DNAs can be analyzed
the effect of guanines in the relatively short probe
DNA may be negligible. Ihara et al. [97] intro-
duced the reporter probe (RP) labeled with an
electroactive ferrocenyl marker. The requirement
that the RP has to be bound to the target DNA at
the site close to the electrode surface represents
limitation of this interesting technique. There is
no doubt that this promising approach will be
further improved and applied in the development
of the DNA hybridization sensors. Interesting
approaches of the Japanese authors based on
ferrocenyl labeled ODNs and threading intercala-
tors were recently reviewed by Takenaka [100].
Using a.c. impedance measurement, Hason et
al. [93] show that in addition to the threading
intercalators the natural bisintercalator echino-
mycin is another redox indicator showing higher
affinity for dsDNA at the electrode surface than
the simple intercalators which have been predomi-
nantly used as redox indicators in DNA hy-
bridization (reviewed in Ref. [2]). The results of
Hason et al. [93] are in agreement with the previ-
ously published CV measurements of echinomycin
binding to DNA [3,101].
The above papers reporting on progress in the
development of the electrochemical DNA hy-
bridization sensors are concerned with hybridiza-
tion and detection of the hybridization event at a
815
(single) surface of a solid electrode. A new ap-
proach is shown in this issue for the first time
based on hybridization at one surface (surface H,
optimized for this purpose) and electrochemical
detection at another surface (the detection elec-
trode, DE). This new system has many advan-
tages as detailed in [73,94,102]. One of them is
that electrodes poorly suitable for DNA hy-
bridization, but well suited for the detection, can
be used as DEs. We took this advantage to de-
velop a label-free detection of the DNA hy-
bridization by means of the cathodic stripping
voltammetry (CSV) of adenine released from
DNA by acid treatment. Adenine, forming spar-
ingly soluble compounds with the electrode mer-
cury, can be determined by CSV at ppb level [16];
a 20 ml volume is sufficient for the analysis [73].
Using superparamagnetic Dynabeads Oligo
(dT)25 (DBT, with covalently bound probe DNA)
as surface H and HMDE as DE we were able to
detect long target ODNs (67 nucleotides),
polyadenylic acid, mRNA from total RNA and
DNA PCR product (over 220 nucleotides). Our
work might be considered as an early develop-
ment showing new opportunities for the mercury
and amalgam electrodes in the DNA hybridiza-
tion sensors. DBT in combination with chemical
modification of target DNA (making the DNA
immunogenic) was used for electrochemical im-
munoassay [102]. In this case the enzyme was
bound to the monoclonal antibody against DNA
modified by Os-bipy complex [103]. Electroinac-
tive 1-naphthyl phosphate used as a substrate was
turned by the enzyme into 1-naphthol which was
determined at pyrolytic graphite electrode. 67-mer
ODN as well as a DNA PCR product ( B 220
base pairs) were detected at high sensitivities.
Using DBT the analysis can be performed with
samples containing high excess of noncomplemen-
tary DNA; this is particularly important for fur-
ther development of the DNA sensors.
In this issue J. Wang et al. [94] used another
type of the paramagnetic beads (covered with
streptavidin to which biotinylated ODN probe
was bound). Biotin-labeled 19-mer was used as a
target DNA; and streptavidin-labeled alkaline
phosphatase was bound to the biotinylated target
DNA. The same substrate/product system as
816 E. Paleček / Talanta 56 (2002) 809–819
above and disposable screen-printed carbon elec-
trodes were used. The detection limit of 0.5 ng of
target DNA in 50 ml volume was calculated from
the measurements of 100 ng of target DNA per
ml. In another paper, Wang et al. [104] used the
same beads in combination with gold nanoparticle
tags and precipitation of silver on these nano-
particles and obtained equally good detection
limit of the biotin labeled ODN 19-mer target
hybridization.
Kizek et al. [90] show in this issue that ODNs
end-labeled with an SH group can be easily
covalently bound to the surface of HMDE and
signals due to sulfur and base residues can be
measured. Their paper together with our prelimi-
nary data (E. Palecek, L. Havran, R. Kizek,
unpublished) suggest that with thiolated ODNs
mercury electrode might serve not only for the
DNA detection but also for DNA hybridization.
9. Future
9.1. Nucleic acid electrochemistry and molecular
biology
If DNA sensors become commercially available
and widely applied, the future of the NA electro-
chemistry will be bright; huge basic research will
be necessary to improve the DNA hybridization
sensor technologies. Optimization of electrodes
and combination of the electrochemical detection
with multiple sample analysis, including microar-
rays and manipulation of the samples using mi-
crofludic systems represent only a fraction of
future tasks oriented toward the development of a
versatile and inexpensive DNA hybridization sen-
sor devices. But even if the DNA sensors are not
considered increasing interest in the DNA electro-
chemistry can be expected. Such expectations are
predicated by the growing importance of the
molecular genetics and molecular medicine on one
hand and with the principles and versatility of the
electrochemical analysis on the other hand. Com-
bination of electrodes with modern visualization
techniques such as scanning probe microscopies
appears particularly interesting.
9.2. Interactions of nucleic acids with proteins
Interactions of NAs with proteins and peptides
is of crucial importance in many living processes.
These interactions are intensively studied by vari-
ous biochemical and molecular biological meth-
ods but electrochemical analysis has not yet been
used for this purpose. This is surprising because in
addition to NAs also proteins produce faradaic
and catalytic electrochemical signals. First reports
about electroactivity of proteins were published
about 70 years ago with the mercury dropping
electrode [105–107] and about 25 years later solid
electrodes entered the field, e.g. [108]. Recent im-
provements in the sensitivity of the protein analy-
sis [60,61,109] is promising and increases the
chance for success of the future electrochemical
studies the DNA-protein interactions.
The highest sensitivity of the protein analysis
has recently been reached with the constant cur-
rent chronopotentiometric stripping analysis
(CPSA). Its sensitivity is by 2–3 orders of magni-
tude higher than that of previously used polaro-
graphic or voltammetric methods and it appears
very attractive for the analysis of proteins. CPSA
signals of proteins and peptides at carbon elec-
trodes are based on the oxidation of tyrosine and
tryptophan residues [61,110]. At the mercury elec-
trodes peptides and proteins produce CPSA ‘peak
H’ [61,90] at highly negative potentials (close to
− 1.7 V against SCE). This peak was recently
applied for the determination of metallothioneins
at femtomol level [111].
Electrochemical methods are able to recognize
native and denatured conformations of DNA and
at least of some proteins. Our first results [112] of
the analysis of the tumor suppressor protein p53
[113] suggest that electrochemistry can be applied
not only to the studies of peptides and simple
proteins but also to studies of complex proteins.
We proposed a method for the determination of
traces of glutathione-S-transferase (p53 is fre-
quently isolated as a fusion protein with this
enzyme) in p53 protein samples [112]. Application
of osmium tetroxide complexes to modify and
determine peptides and proteins is another inter-
esting topic. Using DPV we have been able to
determine peptide hormones at subnanomolar
 E. Paleček / Talanta 56 (2002) 809–819
concentrations [114]. Our preliminary data sug-
gest that Os,L complexes can be applied also for
analysis of proteins, including p53 (E. Palecek, M.
Brázdová, S. Billova, unpublished).
Due to the recent progress in the analysis of
nucleic acids and proteins the electrochemical
methods appear ready for application to se-
quence-specific and nonspecific interactions of
proteins with DNA. Such application may involve
not only DNA-protein interactions in solution but
also interactions with one component immobilized
at the surface and the other in solution. The
number of proteins known to interact with DNA
is large, including antibodies, oncoproteins, tran-
scription factors, etc. The problem is that for
correct application of electrochemistry in this
field, the electrochemist must know a considerable
amount of biology and the biologist has to learn
some electrochemistry. Close collaboration be-
tween electrochemists, biochemists, molecular bi-
ologists and biotechnologists may be the best
solution of the dilemma. It is hard to imagine any
substantial progress in nucleic acid electrochem-
istry without such collaborations.
Acknowledgements
I am indebted to Drs M. Heyrovský, M. Fojta,
J. Janata J. Slotova and J. Wang for critical
reading of the manuscript. This work was sup-
ported by grant of the Grant Agency of the Czech
Republic No. 204/97/K084 and Z 5004920 of the
Academy of Sciences of the Czech Republic.
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