Electropolymerization of intercalator-grafted conducting polymer for direct and amplified DNA detection

Article abstract of DOI:10.1039/c0cc03698f

Efficient DNA intercalators comprising naphthalene diimide grafted with ethylenedioxythiophene monomer were successfully designed, synthesized and applied to mediate/promote conducting polymer growth electrochemically on biosensor electrodes for direct and amplified DNA detection.

Full text of DOI:10.1039/c0cc03698f

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Electropolymerization of intercalator-grafted conducting polymer
for direct and amplified DNA detectionw
a
a
Natalia C. Tansil,z Eric Assen B. Kantchev,z Zhiqiang Gaoa and Hsiao-hua Yu*b
Received 7th September 2010, Accepted 29th October 2010
DOI: 10.1039/c0cc03698f
Efficient DNA intercalators comprising naphthalene diimide
grafted with ethylenedioxythiophene monomer were successfully
designed, synthesized and applied to mediate/promote conducting
polymer growth electrochemically on biosensor electrodes for
direct and amplified DNA detection.
to improve the solubility of the compounds in aqueous
medium. Condensation of excess EDOT–EG4–NH2 with
1,4,5,8-naphthalene tetracarboxylic dianhydride yielded the
symmetrical EDOT–ND–EDOT.11 For the synthesis of
asymmetric EDOT–ND–Os, the ND precursor was first
reacted with 1 equivalent of 1-(3-aminopropyl)imidazole to
form mono-imide.12 Subsequently, the intermediate was reacted
with EDOT–EG3–NH2 to yield ND with asymmetrical
imidazole and EDOT grafts (EDOT–ND–Im).13 Complexation
between this compound and Os(bpy)2Cl2 (bpy = bipyridine)
was conducted in refluxing ethylene glycol.
Intercalating organic/inorganic compounds that bind selectively
and reversibly to double-stranded DNA (dsDNA) have
demonstrated applications for antitumor drugs, DNA probes
and gene delivery vectors.1 Redox-active and fluorescent
intercalators are employed as indicators for DNA hybridization
to avoid labeling of the target DNA.2 Complementary DNA
targets are detected by measuring optical/electrochemical
signals of these intercalators. However, most of these methods
are limited by low signal intensity and poor signal/noise ratio.
Recently, several reports have demonstrated sensitive DNA
1
In addition to H and 13C NMR and HR-MS, the target
compounds were characterized by UV-Vis spectroscopy
(Fig. 1A) and cyclic voltammetry (Fig. 1B). EDOT–ND–Os
exhibited absorption bands from Os(bpy)22+ (lmax = 296 nm)
and ND (lmax = 359 and 379 nm), whereas EDOT–ND–EDOT
only displayed absorption bands arising from ND. EDOT–
ND–Os displayed redox wave from Os2+/Os3+ (E1/2 = 0.17 V)
upon oxidation, and redox waves from the p-conjugated
system of ND (E1/2 = ꢀ1.04 and ꢀ0.63 V) upon reduction.14
In intercalation, insertion of the planar aromatic ring
between dsDNA base pairs would result in hypochromism
and red shifts in UV-Vis absorption spectra. As shown in
Fig. 1C, addition of salmon sperm DNA to a solution of
EDOT–ND–Os at a DNA base pair/EDOT–ND–Os ratio of
3.0 resulted in 40% decrease in ND absorption bands at
362 and 383 nm. These bands were also red-shifted by B3 nm,
consistent with the previous reports on ND with aliphatic
detection with
a greatly amplified signal generated by
nanoparticle-mediated3 or enzyme-catalyzed4 material growth
on sandwich DNA assay (capture probe/target DNA/detection
probe). Herein we present a unique molecular approach for
intercalator-mediated polymer growth by combining this
signal amplification method with the advantageous use of
intercalators for DNA detection. This method is based on a
designed dual functional molecule with both intercalator and
conductive monomer building blocks that facilitates the
electrochemical growth of conducting polymer on an electrode
with hybridized DNA.
Naphthalenediimide (ND) intercalators5 conjugated to
redox-active moieties have been applied for electrochemical
DNA detection.6 On the other hand, conducting polymers
derived from EDOT are among the most promising conductive
materials due to their high conductivity, long-term stability
and structure versatility.7,8 We synthesized two ND-derivatized
molecules with EDOT building block symmetrically
(EDOT–ND–EDOT) and unsymmetrically (EDOT–ND–Os)
as shown in Scheme 1.
2+
tertiary amine side-chains5,15 or symmetrical Os(bpy)2
complex.6 In contrast, limited hypochromism and red-shifts
were observed for EDOT–ND–EDOT (Fig. 1D), presumably
due to limited binding capacity arising from the lack of
positive charges. In most DNA detection, the concentration
of capture probe–target DNA hybrid is so low that intercalation
is unlikely to occur by pure diffusion. For positively charged
EDOT–ND–Os, favorable binding occurred between the
polyanionic DNA and the cationic intercalator due to the
additional electrostatic interactions, leading to stronger
intercalation. Intercalative properties of EDOT–ND–Os were
further studied by a competitive displacement assay with
thiazole orange dye. The binding constant was estimated as
8 ꢁ 105 Mꢀ1 with dsDNA.
Starting from hydroxymethyl-functionalized EDOT,9 we
prepared
a key EDOT intermediate containing amino
functional group with oligoethylene glycol linkers
(EDOT–EGn–NH2; n = 3 or 4).10 These linkers were introduced
a Institute of Bioengineering and Nanotechnology, 31 Biopolis Way,
The Nanos #04-01 Singapore 138669
Both EDOT–ND–EDOT and EDOT–ND–Os electro-
polymerized to form homopolymer or copolymers in either
organic or aqueous microemulsion electrolyte solutions. Cyclic
voltammograms of copolymer films of EDOT–ND–Os/
EDOT–OH clearly displayed reversible peaks (E1/2 = 0.2 V)
from Os2+/Os3+ redox centers. The peaks were superimposed
on the broad wave of redox activities from the PEDOT
backbone, confirming the formation of the copolymer.
b Yu Initiative Research Unit, RIKEN Advanced Science Institute,
2-1 Hirosawa, Wako, Saitama 3510198, Japan.
E-mail: bruceyu@riken.jp; Fax: +81 48 462 1659;
Tel: +81 48 467 9512
w Electronic supplementary information (ESI) available: Details of
synthetic procedures, electrochemical experiments and DNA
detection. See DOI: 10.1039/c0cc03698f
z Current address: Institute of Materials Research and Engineering,
3 Research Link, Singapore 117602.
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 1533–1535 1533

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Scheme 1 Synthesis of EDOT-grafted naphthalenediimide intercalators.
higher peak current was obtained for 20 pM of complementary
target as compared to that of a blank experiment without
target and for controls of 100 pM of non-complementary
target (50-GGTTCGTTGTCTGAGTTT) and 2-base mismatched
target (50-CCAAGGAACAGAGTCAAA). However, the signal
differences were limited, even after background correction.
Detection of 20 pM target using this method is not satisfactory.
Even at 100 pM target concentration, the current signal of the
complementary target was only B4 fold of the signal from the
non-complementary target (Fig. 2A).
One advantageous feature of these new intercalators was
their capability to electropolymerize to form PEDOT films as
described earlier. As shown in Fig. 2A, direct electrochemical
detection of electroactive intercalators is not sensitive enough
and could only lead to a detection limit of around 100 pM and
a very low current output (B10 nA) even with a more sensitive
square wave voltammetric method. We would like to amplify
the electrochemical signal output using the greater electro-
activity of PEDOT films, which could lead to easier and
cheaper instrumentation. The biosensor electrode was first
placed in aqueous electrolyte solution (0.1 M of LiClO4),
and subjected to cyclic potential between ꢀ0.2 and 1.0 V.
For electrodes hybridized with complementary target, a greater
Fig. 1 (A) UV-Vis absorption spectra and (B) cyclic voltammograms
of EDOT–ND–Os (—), EDOT–ND–EDOT (- - -), and Os(bpy)2Cl2
(ꢂ ꢂ ꢂ). The UV-Vis spectra were obtained in ethanol solution. Cyclic
voltammograms were measured in 0.1 M of nBu4NPF6/CH3CN (for
EDOT–ND–Os and EDOT–ND–EDOT) and phosphate buffered
saline (PBS) (for Os(bpy)2Cl2) at a scan rate of 100 mV sꢀ1. UV-Vis
absorption spectra of 25 mM of (C) EDOT–ND–Os and
(D) EDOT–ND–EDOT in PBS buffer solution containing 0, 25, 50,
and 75 mM (from top to bottom) of salmon sperm DNA.
The concentration of DNA is based on base pairs.
For DNA detection, thiol-functionalized peptide nucleic acid
(PNA) capture probes (50-TTTGAGTCTGTTGCTTGG) and
mercaptoundecanol were self-assembled on gold electrode
surface.16 PNA probes were used in order to reduce the non-
specific binding of cationic EDOT–ND–Os and enhance the
hybridization efficiency by eliminating the anionic repulsion
between DNA targets and DNA probes. The probe sequence
was designed to target the N1 gene of avian flu virus. After
hybridization with the complementary target (50-CCAAGCAA-
CAGACTCAAA), EDOT–ND–Os intercalation and washing,
redox activity of Os2+/Os3+ was observed electrochemically.
Using the more sensitive square-wave voltammetric method, a
Fig. 2 (A) Peak current from square-wave voltammograms of
EDOT–ND–Os bound to DNA biosensor electrodes at 0.14 V. (B)
Voltammetric current from cyclic voltammograms of PEDOTs formed
on assembled biosensor electrodes at 0.3 V (oxidation). The electrodes
were hybridized with 100 pM of complementary target (white), 20 pM
of complementary target (light grey), 20 pM of two-base mismatched
target (dark grey) and 100 pM of non-complementary target (black).
Base line signal (no target) was subtracted from the experimental data.
c
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increase of current was obtained between the initial and
subsequent cycles due to the formation of oligomers/polymers
from the surface-bound EDOT–ND–Os. We found a greater
difference between the electrochemical signals of the experi-
mental and control biosensor electrodes. The oligomers/
polymers formed at this stage would serve as ‘nuclei’ for
subsequent growth of PEDOT.
Notes and references
1 DNA and RNA Binders: From Small Molecules to Drugs,
ed. C. Demeunynck, C. Bailly and W. D. Wilson, Wiley-VCH,
Weinheim, 2003.
2 E. M. Boon, D. M. Ceres, T. G. Drummond, M. G. Hill and
J. K. Barton, Nat. Biotechnol., 2000, 18, 1096; F. Patolsky, E. Katz
and I. Willner, Angew. Chem., Int. Ed., 2002, 41, 3398; Q.-g. Chen,
D.-h. Li, H.-h. Yang, Q.-z. Zhu and J.-g. Xu, Analyst, 1999, 124,
901; G. Marrazza, I. Chianella and M. Mascini, Anal. Chim. Acta,
1999, 387, 297; J. Wang, X. Cai, G. Rivas, H. Shiraishi, P. A.
M. Farias and N. Dontha, Anal. Chem., 1996, 68, 2629; B. J. Taft,
M. A. Lapierre-Delvin and S. O. Kelley, Chem. Commun., 2006, 962.
3 T. A. Taton, C. A. Mirkin and R. L. Letsinger, Science, 2000, 289,
1757; S.-J. Park, T. A. Taton and C. A. Mirkin, Science, 2002, 295, 651.
4 Z. Gao, S. Rafea and L. H. Lim, Adv. Mater., 2007, 19, 602;
Y. Fan, X. T. Chen, A. D. Trigg, C. H. Tung and J. M. J. Kong,
J. Am. Chem. Soc., 2007, 129, 5437.
5 S. F. Yen, E. J. Gabbay and W. D. Wilson, Biochemistry, 1982, 21,
2070; F. A. Tanious, S. F. Yen and W. D. Wilson, Biochemistry,
1991, 30, 1813.
6 S. Takenaka, K. Yamashita, M. Takagi, Y. Uto and H. Kondo,
Anal. Chem., 2000, 72, 1334; N. C. Tansil, H. Xie, F. Xie and
Z. Gao, Anal. Chem., 2005, 77, 126; N. C. Tansil, F. Xie, H. Xie
and Z. Gao, Chem. Commun., 2005, 1064.
7 L. Groenendaal, G. Zotti, P.-H. Aubert, S. M. Waybright and
J. R. Reynolds, Adv. Mater., 2003, 15, 855; J. Roncali,
P. Blanchard and P. Frere, J. Mater. Chem., 2005, 15, 1589.
8 For recent examples, see: T. Hatano, A.-H. Bae, M. Takeuchi,
N. Fujita, K. Kaneko, H. Ihara, M. Takafuji and S. Shinkai,
Angew. Chem., Int. Ed., 2004, 43, 465; E. Mohamed Ali, E. A.
B. Kantchev, H.-h. Yu and J. Y. Ying, Macromolecules, 2007, 40,
6025; J. F. Hulvat and S. I. Stupp, Angew. Chem., Int. Ed., 2003,
42, 778; J. Jang, M. Chang and H. Yoon, Adv. Mater., 2005, 17,
1616; F. Mouffouk and S. J. Higgins, Electrochem. Commun., 2006,
8, 15; S.-C. Luo, H. Xie, N. Chen and H.-h. Yu, ACS Appl. Mater.
Interfaces, 2009, 1, 1414; H. Xie, S.-C. Luo and H.-h. Yu, Small,
2009, 5, 2611; S.-C. Luo, S. S. Liour and H.-h. Yu, Chem.
Commun., 2010, 46, 4731; S.-C. Luo, J. Jiang, S. S. Liour,
S. Gao, J. Y. Ying and H.-h. Yu, Chem. Commun., 2009, 2664.
9 S. Lima, P. Schottland, S. Sadki and C. Chevrot, Synth. Met.,
1998, 93, 33; S. Akoudad and J. Roncali, Electrochem. Commun.,
2000, 2, 72.
To further enhance the signal difference, the electrodes were
placed in an aqueous electrolyte solution containing 5 mM of
EDOT–OH. Previous reports on electrochemical growth of
conducting polymers have shown that the polymer would
form at a much lower potential after the initial layer of
polymer has been deposited.9 This was likely due to the lower
over-potential required from improved electron transfer and
alternative stepwise polymer propagation steps besides the
original radical–radical coupling.17 After fine-tuning the elec-
trochemical polymer growth condition, it was found that
amperometric polymer growth at 0.9 V for 120 s was optimal.
At this controlled condition, polymer growth is severely
limited in the absence of any nucleating sites. This results in
a lower background, and thus potentially lower detection
limit. This improved detection method is summarized in
Scheme S1 (ESIw). As shown in Fig. 2B, greater signal
difference was observed between complementary and
non-complementary targets as compared to the electro-
chemical signal previously observed from Os2+/Os3+ even
with a less sensitive cyclic voltammetric method (Fig. S1,
ESIw). Applying higher potentials or prolonging the polymeri-
zation time would result in
a significant increase of
polymer growth on the control experiments, effectively
reducing the contrast. A much higher contrast of current
outputs was obtained between biosensors with complementary
and non-complementary DNA targets. This enhanced
contrast was >100-fold improvement compared to the
contrast obtained from the square-wave method applied in
Fig. 2A, and detection of 20 pM target can be satisfactorily
achieved. Using the same method with various concentrations
of complementary target, a calibration plot was obtained as
presented in Fig. S3 (ESIw).
10 M. Besbes, G. Trippe
I. F. Perepichka, A. Derdour, A. Gorgues, M. Salle
J. Roncali, Adv. Mater., 2001, 13, 1249; G. Trippe, F. Le Derf,
J. Lyskawa, M. Mazari, J. Roncali, A. Gorgues, E. Levillain and
M. Salle, Chem.–Eur. J., 2004, 10, 6497.
´
, E. Levillain, M. Mazari, F. Le Derf,
´
and
´
´
11 H. E. Katz, J. Johnson, A. J. Lovinger and W. Li, J. Am. Chem.
Soc., 2000, 122, 7787; N. Rahe, C. Rinn and T. Carell,
Chem. Commun., 2003, 2120.
In conclusion, a bi-functional molecule containing both an
efficient dsDNA intercalator (ND) and a monomer unit of
thiophene-based conducting polymer (EDOT) has been
designed and synthesized. This molecule provides a unique
approach to promote electrochemical growth of conducting
polymer on dsDNA-immobilized electrode for sensitive DNA
detection. Combining with cheaper and portable electrical
instruments, the low detection limit and large current output
would be potentially useful for point-of-care diagnostics.
The authors thank Dr Lisa F. P. Ng (Singapore Immunology
Network) for helpful discussions on DNA probe design. This
work is funded by the Institute of Bioengineering and Nano-
technology (Biomedical Research Council, Agency for
Science, Technology and Research, Singapore) and RIKEN
Advanced Science Institute.
12 I. Tabakovic, L. L. Miller, R. G. Duan, D. C. Tully and
D. A. Tomalia, Chem. Mater., 1997, 9, 736.
13 EDOT compounds with similar structures have been recently
´
published, see J. L. Segura, R. Gomez, E. Reinold and P. Bauerle,
¨
Org. Lett., 2005, 7, 2345; J. L. Segura, R. Go
´
E. Reinold and P. Bauerle, Chem. Mater., 2006, 18, 2834.
mez, R. Blanco,
¨
14 All potentials in the report are referenced to Ag/AgCl electrode.
A standard three-electrode set-up was used for all electrochemical
studies with Pt button electrode as the working electrode, Pt coil as
the counter electrode, and Ag/Ag+ (non-aqueous solution) or
Ag/AgCl (aqueous solution) as the reference electrode.
15 D. W. Dixon, N. B. Thornton, V. Steullet and T. Netzel,
Inorg. Chem., 1999, 38, 5526.
16 Cationic polythiophenes have been applied for electrochemical
DNA detection purely based on electrostatic interactions, see:
F. Le Floch, H.-A. Ho, P. Harding-Lepage, M. Bedard,
´
R. Neagu-Plesu and M. Leclerc, Adv. Mater., 2005, 17, 1251.
17 J. Roncali, Chem. Rev., 1992, 92, 711.
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 1533–1535 1535