We report on the design and development of a novel label-free DNA sensor based on conducting poly(3,4-ethylenedioxythiophene) for the direct detection and quantification of target ssDNA

Contractor, Aliasgar Q.

Gokhale, Rajesh S.

Krishnamoorthy, K.

Kumar, Anil

English

Publications of the IAS Fellows

Novel label-free DNA sensors based on
poly(3,4-ethylenedioxythiophene)†
K. Krishnamoorthy,a Rajesh S. Gokhale,b Aliasgar Q. Contractora and Anil Kumar*a
a Chemistry Department, Indian Institute of Technology Bombay, Mumbai 400076, India.
E-mail: anilkumar@iitb.ac.in
b National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India
Received (in Cambridge, UK) 23rd December 2003, Accepted 6th February 2004
First published as an Advance Article on the web 24th February 2004
We report on the design and development of a novel label-free
DNA sensor based on conducting poly(3,4-ethylenedioxythio-
phene) for the direct detection and quantification of target
ssDNA.
In this age of molecular genetics a lot of research efforts are
directed towards the understanding of DNA and RNA sequences in
order to diagnose, prevent, and treat many human diseases. Simple,
economical and efficient detection of various nucleotides se-
quences is also of prime importance for applications such as
forensic medicine, rapid detection of biological warfare agents, and
environmental testing.1 The basic principle for the detection of
DNA relies on the detection of duplex formation between a single
strand DNA (ssDNA), which is part of the sensor, with that of the
target DNA having a sequence that is exactly complementary.2
In an electrochemical DNA hybridization detector, a short
ssDNA is usually immobilized on a transducer to create a DNA
recognition element. Hybrid formation is then translated into an
electrical, analytically useful signal. Formation of the DNA
recognition layer and the ability to detect the duplex formation
determines the specificity and the sensitivity of the device.
Conjugated polymer matrices offer extraordinary potential to act as
transducers for detecting such duplex formation because the
electrical, optical, and electrochemical properties are strongly
affected by relatively small perturbations. As a result of this
sensitivity, conjugated polymers have been used for various
chemical and biological sensors.3 The main advantages of these
devices are their low-cost, simple design, small dimensions, and
low power requirements. Two distinct approaches have been used
for the immobilization of ssDNA into conducting polymer
matrices. The first approach is based on the electrostatic interaction
wherein the ssDNA was immobilized after the polymerization by
keeping the polymer in an oxidized state.4,5 This restricts the
amount of loading and the probe leaches out during the meaurement
which then results in poor sensitivities. The second approach is
based on the incorporation of ssDNA during the polymerization.
This can be achieved either by the physical entrapment of ssDNA
during the electropolymerization6 or by using a monomer contain-
ing ssDNA as a side chain.7 Although more stable and sensitive
DNA sensors can be fabricated using the ssDNA modified
monomers, synthetic difficulties often restrict its potential.
In principle, DNA sensors based on the direct physical
entrapment of the ssDNA into the conjugated polymer matrix have
the advantage of ease of synthesis and can be used as label-free
detection of target DNA. Therefore, this method has the potential
for commercialization, provided the specificity and the sensitivity
are significant. Interestingly, there is only one report in the
literature where an attempt has been made in this direction using
polypyrrole films as transducers, wherein oligonucleotides contain-
ing either adenine or guanine acted as probes.6 Although the sensor
response was found to be linear when adenine was used as a probe,
the change in currents was very small (from 1 nA to 4 nA) along
with a very small linear range (1–7 3 1026 g). The linear response
was even smaller for a guanine probe. Surprisingly, these sensors
responded to non-complementary targets as well and this has been
attributed to the redox behaviour of polypyrrole in the presence of
different counter ions. It is clear from the above discusssion that a
label-free DNA sensor based on a conducting polymer which is
specific to the complementary target and detect various concentra-
tions with a linear range has not yet been reported.
Here we report on the development of a novel design for the
fabrication of biosensors for DNA detection. The present sensor has
many salient features such as: no labelling or modification of the
ssDNA or the transducing matrix, ease of fabrication, better
sensitivity, higher detection range, detection of small segments in a
longer oligonucleotide and faster response time. In order to
fabricate a DNA sensor, PEDOT was used as an immobilization
matrix-cum-physicochemical transducer because PEDOT is highly
stable, can be synthesized and used at physiological pH and its
conductivity does not change significantly with changes in counter
ions.8 The concept of the present sensor is based on the change in
the conformation of the polymer due to the formation of a DNA
duplex. This is manifested macroscopically in terms of changes in
the conductivity of the polymer. This concept has been explored by
us successfully in the past for the design and fabrication of various
sensors.9
PEDOT microtubules were synthesized from EDOT (0.01 M)
and 0.1 M KCl dissolved in pH 8 phosphate buffer in the pores of
a gold coated polycarbonate membrane by cycling the potential
between 20.2 V and 1.3 V vs. SCE with a scan rate of 50 mV s21.10
Polymerization started at one side of the electrode, grew through
the pore and reached the other side of the pore. This eventually
connects the two gold electrodes on the opposite sides of the
membrane. The PEDOT microtubules were found to be stable in pH
8 phosphate buffer solution. The DNA sensors were fabricated by
electropolymerization from EDOT solution as mentioned above but
in the presence of probe ssDNA. In order to study the effect of
ssDNA length, sensors with 20mer, 10mer and 5mer ssDNA (Table
1) were prepared. Irrespective of the number of bases in the
oligonucleotide probe the redox behavior of PEDOT remained the
same. The sensor measurements were carried out by exposing the
device to various concentrations of complementary ssDNA in pH 8
phosphate buffer solution. The response is represented by DR/R0
where DR = R2 R0; R0 is the resistance of the sensor without any
substrate and R is the resistance of the sensor in the presence of the
substrate. The response of the devices was measured following the
reported experimental technique.10 The sensor prepared with 20 mg
ml21 of ssDNA (20mer A) was exposed to various concentrations
of complementary ssDNA (A1). The sensor measurements were
† Electronic Supplementary Information (ESI) available: Experimental
details and sensor response for various sensors listed in Table 2. Cyclic
voltammograms confirming the increase in resistance due to the duplex
formation between the probe and target DNA in conducting polymer matrix.
See http://www.rsc.org/suppdata/cc/b3/b316794a/
Table 1 Oligonucleotide sequences
ssDNA Sequence
A 5A-GGACATGTTGCCGAAGCG GG-3A
B 3A-GGGCGAAGCC-5A
C 5A-CCC GC-3A
A1 3ACCTGTACAACGGCTTCGCCC-5A
B1 5A-CCC GCT TCG G-3A
C1 3A-GGG CG-5A
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a
8 2 0 C h e m . C o m m u n . , 2 0 0 4 , 8 2 0 – 8 2 1
done at a gate potential of +0.8 V because the sensor response was
found to be highest at this potential. The linear range was observed
from 8 3 1028 to 1 3 1025 g ml21 (Fig. 1). In order to prove that
the sensor response is due to the DNA duplex formation, a control
experiment was done using PEDOT synthesized in the absence of
oligonucleotide probe. This sensor showed negligible response
when it was exposed to various concentrations of ssDNA (A1) (Fig.
1). In order to prove that the sensor response is a result of the duplex
formation between the probe and the target ssDNA in the
conducting polymer, the electrode was kept in ethidium bromide
for 30 min after the sensor measurement. The same experiment was
done for the control experiment as well. Both the electrodes were
taken from ethidium bromide and kept in Tris–EDTA solution for
1 h. Fluorescence spectra were recorded by exciting the solution at
254 nm. The solution obtained from the electrode containing the
immobilized probe ssDNA emits at 314 and 372 nm (Fig. 2).
However, the solution from the control experiment emits at the
same wavelength but the peaks are very weak. From this
experiment it can be concluded that the DNA duplex has formed in
the conducting polymer between the probe and the complementary
ssDNA.
In order to study the effect of ssDNA length, sensors were
fabricated using 20 mg ml21 of 10mer of ssDNA (B). This sensor
was exposed to various concentrations of complementary ssDNA
(B1) and the linear range was observed from 6 3 1027 to 1 3 1025
g ml21 (Table 2). Note that the linear range as well as the sensor
response (450 times) was found to be more in the case of 20mer,
which is due to the large conformational changes upon duplex
formation. Furthermore, the sensor based on 10mer ssDNA (B) was
exposed to various concentrations of noncomplementary 20mer
strand (A) in order to confirm the specificity. No significant
changes were observed in sensor response indicating that the sensor
response is specific to the complementary strand. The true potential
of these sensors will be if a smaller probe strand can be used to
detect a longer target strand having the complementary sequence
anywhere along the length and vice versa. The sensor response for
these types of devices is also listed in Table 2. In these experiments
the resistance of the film has increased upon increasing the
concentration of the complementary strand in the solution. The
increase in resistance of the film upon duplex formation was
confirmed by cyclic voltammetry where a decrease in redox charge
was observed upon duplex formation.
In conclusion, we have shown that the present DNA sensor is
novel in many respects such as: a) this is a label-free DNA sensor
which can be fabricated easily; b) it can detect concentrations as
low as 80 3 1029 g ml21; c) a transducer immobilized with a 5mer
can be used to detect a longer strand. The lower limit for the
detection can be further improved by increasing the loading of the
probe ssDNA and work is in progress in this direction.
We would like to thank MHRD and DST India for financial
help.
Notes and references
1 J. Wang, Chem. Eur. J., 1999, 5, 1681.
2 R. Chakrabarti and A. M. Klibanov, J. Am. Chem. Soc., 2003, 125,
12531; L. He, M. D. Musick, S. R. Nicewarner, F. G. Salinas, S. J.
Benkovic, M. J. Natan and C. D. Keating, J. Am. Chem. Soc., 2000, 122,
9071; J. J. Storhoff, R. Elghanian, R. C. Mucic, C. A. Mirkin and R. L.
Letsinger, J. Am. Chem. Soc., 1998, 120, 1959; Y. W. Cao, R. Jin and
C. A. Mirkin, J. Am. Chem. Soc., 2001, 123, 7961; B. S. Gaylord, A. J.
Heeger and G. C. Bazan, J. Am. Chem. Soc., 2003, 125, 896; B. S.
Gaylord, A. J. Heeger and G. C. Bazan, Proc. Natl. Acad. Sci., USA,
2002, 99, 10954; H.-A. Ho, M. Boissinot, M. G. Bergeron, G. Corbeil,
K. Dore, D. Boudreau and M. Leclerc, Angew. Chem., Int. Ed., 2002, 41,
1548; C. Fan, K. W. Plaxco and A. J. Heeger, Proc. Natl. Acad. Sci.,
USA, 2003, 100, 9134.
3 D. T. Mcqquade, A. E. Pullen and T. M. Swager, Chem. Rev., 2000, 100,
2537; G. G. Wallace and L. A. P. Kane-Macguire, Adv. Mater., 2002,
14, 953; J. Janata and M. Josowicz, Acc. Chem. Res., 1998, 31, 241.
4 D. S. Minehan, K. A. Marx and S. K. Tripathy, J. Macromol. Sci., Pure
Appl. Chem., 2001, A38(12), 1245.
5 D. S. Minchan, K. A. Marx and S. K. Tripathy, Macromolecules, 1994,
27, 777; L. A. Thompson, K. Kowalik, M. Josowicz and J. Janata, J. Am.
Chem. Soc., 2003, 125, 325.
6 J. Wang, M. Jiang, A. Fortes and B. Mukherjee, Anal. Chim. Acta, 1999,
402, 7.
7 H. Korri-Youssoufi, F. Garnier, P. Srivastava, P. Godillot and A.
Yassar, J. Am. Chem. Soc., 1997, 119, 7388.
8 P.-H. Aubert, L. Groenendaal, F. Louwet, L. Lutsen, D. Vanderzande
and G. Zotti, Synth. Met., 2002, 126, 193.
9 M. Kanungo, D. N. Srivastava, A. Kumar and A. Q. Contractor, Chem.
Commun., 2002, 680; R. B. Dabke, G. D. Singh, A. Dhanabalan, R. Lal
and A. Q. Contractor, Anal. Chem., 1997, 69, 724.
10 M. Kanungo, A. Kumar and A. Q. Contractor, Anal. Chem., 2003, 75,
5673; M. Kanungo, A. Kumar and A. Q. Contractor, J. Electroanal.
Chem., 2002, 528, 46; S. Sukeerthi and A. Q. Contractor, Chem. Mater.,
1998, 10, 2412.
Fig. 1 Sensor response as a function of ssDNA concentration (A1). -,
Complementary strand; < , control experiment (PEDOT device fabricated
in the absence of ssDNA probe).
Fig. 2 Fluroscence spectra of (a) ethidium bromide intercalated dsDNA and
(b) a control experiment where no dsDNA was expected (see text for
details).
Table 2 Detection range for various sensors
Probe ssDNA
Conc.a/
mg ml21 Target ssDNA Detection range/g ml21
A (20mer) 20 A1 (20mer) 80 3 1029–0.01 3 1023
B (10mer) 20 B1 (10mer) 0.6 3 1026–0.01 3 1023
B (10mer) 20 A (20mer) —
C1 (5mer) 10 B1 (10mer) 0.9 3 1026–6 3 1026
C1 (5mer) 10 B (10mer) —
B (10mer) 10 C (5mer) 0.5 3 1026–3 3 1026
a Concentration in the polymerization solutions.
C h e m . C o m m u n . , 2 0 0 4 , 8 2 0 – 8 2 1 8 2 1


Novel label-free DNA sensors based on poly (3, 4-ethylenedioxythiophene)

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Novel label-free DNA sensors based on poly (3, 4-ethylenedioxythiophene)

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