Electrochemical signal modulation in homogeneous solutions using the formation of an inclusion complex between ferrocene and β-cyclodextrin on a DNA scaffold

Article abstract of DOI:10.1039/c1cc15365j

Two DNA conjugates modified with ferrocene and β-cyclodextrin were prepared as a pair of probes that work cooperatively for DNA sensing, in which the electrochemical signal of ferrocene on one probe was significantly "quenched" by the formation of an incl

Full text of DOI:10.1039/c1cc15365j

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Electrochemical signal modulation in homogeneous solutions using the
formation of an inclusion complex between ferrocene and b-cyclodextrin
on a DNA scaffoldw
ab
a
a
a
a
Toshihiro Ihara,* Tsugutoshi Wasano, Ryuta Nakatake, Pelin Arslan, Akika Futamura
and Akinori Jyoa
Received 29th August 2011, Accepted 9th October 2011
DOI: 10.1039/c1cc15365j
Two DNA conjugates modified with ferrocene and b-cyclodextrin
were prepared as a pair of probes that work cooperatively for DNA
sensing, in which the electrochemical signal of ferrocene on one
probe was significantly ‘‘quenched’’ by the formation of an inclusion
complex with b-cyclodextrin of the other probe on the DNA
templates.
This would free the preparation of electrochemical molecular
9
sensors from the need to anchor the molecules on the electrodes.
Here we examined the possibility of electrochemical signal
modulation in a homogeneous solution using the controlled
interaction between two DNA conjugates carrying Fc and
bCyD on one end of synthetic oligodeoxyribonucleotides (ODN).
All the ODNs used in this study are shown in Fig. 1. The
sequences of the two conjugates (Fc-ODN and CyD-ODN )
Various analytical platforms based on electrochemical
responses have been proposed for studying specific interactions
n
n
were designed to be complementary to the adjacent sites of the
1
0
1
,2
targets (target27 and target22N: a part of the TPMT gene ).
A pair of the two conjugates forms a tandem duplex with the
target, in which both modified units are directed convergently
and, hopefully, form a quenched inclusion complex at the
center of the duplexes. This could be regarded as a split probe
involving nucleic acids and/or proteins.
Most of these
methods, however, require one of the participants in the
interactions to be immobilized on the electrode because signal
contrasts arise as the result of changes in the distance between
the electrochemically active probe and the electrode surface
on the targeted events (=change in the local concentration of
the probe on the electrode). Generally, the preparation of
biomolecule-modified electrodes is time-consuming and it is
difficult to obtain reproducible results. We have already
1
1
(
or binary probe) in electrochemistry.
The system of tandem duplex 1 was subjected to UV melting
experiments to examine the interactions between bCyD and Fc
on the DNA framework. All the curves showed biphasic features
with two inflection temperatures caused by successive meltings
3
reported some electrochemical methods for DNA analysis.
1
2
(
Tmax).z The meltings at lower and higher temperatures are
In this series of studies, a gene sensor was developed based on
the interactions between a ferrocene (Fc)-modified DNA
4
probe and a DNA-modified electrode. Fluorimetry for
DNA sensing using b-cyclodextrin (bCyD)-modified DNA
5
,6
has also recently been reported. It is known that bCyD
binds tightly with Fc to form an inclusion complex, and the
electrochemical activity of Fc accommodated in the bCyD
7
cavity is suppressed by being shielded from the bulk solution.
bCyD could therefore be regarded as a ‘‘quencher’’ for
electrochemical signals, similar to an azo-quencher for
8
FRET-based fluorimetry. This means that specific interactions
could be monitored using the electrochemical signals of Fc in
homogeneous solutions, if the inclusion complex is formed
quantitatively in accordance with the relevant interactions.
a
Department of Applied Chemistry and Biochemistry,
Kumamoto University, 2-39-1 Kurokami, Kumamoto 860-8555,
Japan. E-mail: toshi@chem.kumamoto-u.ac.jp;
Tel: +81 96 372-3772
b
Fig. 1 Sequences of synthetic oligodeoxyribonucleic acids used in
this study. Two tandem duplexes (1 and 2) were designed. They consist
of CyD-ODN, Fc-ODN, and the targets (a part of the TPMT gene).
ODN7 and ODN20 are the unmodified ODNs as references.
CREST, JST, Tokyo 102-0075, Japan
w Electronic supplementary information (ESI) available: ODN
conjugates synthesis and results of UV melting, SWVs, and hydro-
dynamic voltammograms. See DOI: 10.1039/c1cc15365j
1
2388 Chem. Commun., 2011, 47, 12388–12390
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+
redox couple (Fc/Fc ) was significantly suppressed only for
the ternary tandem duplex CyD-ODN /target27/Fc-ODN . As
1
1
we expected, the electrochemical activity of Fc in the DNA
conjugate seems to be significantly quenched by shielding with
bCyD on the DNA scaffold. bCyD as an intervening aliphatic
insulator would prevent the Fc from reaching the electrode
directly and would suppress the rate of electron transfer. The
difficulty in accessing the counter anion experienced by ferro-
cenium tightly surrounded by bCyD might be another cause of
the current suppression. Recently, several electrochemical
methods for DNA analysis have been proposed based on
Fig. 2 UV melting curves of tandem duplexes. Solid and broken
curves show the meltings of CyD-ODN /target27/Fc-ODN and
CyD-ODN /target27/ODN20, respectively. Melting experiments were
carried out in 10 mM phosphate buffer solution (pH 7.0) containing
1
4
bCyD–Fc interactions. The contrasts in the magnitude of
the electric currents obtained in the present study are much
higher than those previously reported. This might be the result
of differences in the linker chains such as the length, structure,
and tethering point on bCyD, which would significantly affect
the structure and stability of the inclusion complexes (ESIw).
The effects of the sequence of the target DNAs on the current
1
1
1
5
1
00 mM KCl. The concentrations of the ODN components were all
ꢀ1
.0 mM. The solutions were heated at a rate of 0.5 1C min . Inset
shows the fitting of the melting at lower temperature to the theoretical
curve. Closed and open circles are the experimental data for each
tandem duplex. Red curves are the optimized theoretical curves.
+
based on Fc/Fc were examined using the system of tandem
11a
attributed to those of 7-mer and 20-mer ODNs, respectively.
duplex 2 with the four targets, i.e., target22N carrying one
base displacement. Square-wave voltammograms measured at
Fig. 2 shows the melting curves of the tandem duplexes CyD-
ODN /target27/Fc-ODN and CyD-ODN /target27/ODN20.
4
0 1C are shown in Fig. 3(b). UV melting experiments for these
four tandem duplexes, CyD-ODN /target22N/Fc-ODN , showed
1
1
1
2
2
The Tmax of CyD-ODN
target27/Fc-ODN
1
dissociation from CyD-ODN
1
/
/
that only target22C can form a stable tandem duplex at this
temperature (ESIw). As we expected, only the signal from the
duplex CyD-ODN /target22C/Fc-ODN almost vanished. The
1
was higher than that for CyD-ODN
1
target27/ODN20. The difference in the Tmax values was almost
0 1C. This could be attributed to the stabilization effect of the
2
2
2
current signal increased with increasing temperature. Then,
finally, it gave almost the same magnitude as those of the other
three duplexes at 55 1C (ESIw). This result clearly shows that
the bCyD–Fc inclusion complex formation is controlled by
sequence-specific hybridization of ODNs. Control of the inter-
action is not difficult by taking advantage of the programmability
interaction between bCyD and Fc on the tandem duplex. The
lower temperature regions of both melting curves were fitted
3a,12
with the theoretical equation (Fig. 2, inset).
The thermo-
dynamic parameters obtained and the Tmax values of all the
tandem duplexes are summarized in Table 1. The difference in
o
DG2 for CyD-ODN1 binding with target27/Fc-ODN and
98
1
15
ꢀ
1
and the predictable thermal stability of DNA structures.
with target27/ODN20 (DDG1) was ca. 3.2 kcal mol . This is a
reasonable value as the free energy comes from the formation
7
,13
of a bCyD–Fc inclusion complex.
This corresponds to an
enhancement by ca. 260 times of the binding constant at 25 1C.
This means that the bCyD tethered to the ODN end accommo-
dates the Fc tethered to the neighboring end of another ODN
on the target DNA to form a tight inclusion complex without
significant strain in the structure.
The electrochemical properties of Fc-ODN1 were investi-
gated by cyclic voltammetry using a dual microelectrode.
Fig. 3(a) shows the cyclic voltammograms of Fc-ODN1 in
several states. The current based on the ferrocene/ferrocenium
a
Table 1 Melting temperatures and thermodynamic parameters of
b
duplex formation
Fig. 3 Electrochemical responses of Fc-ODN in several states. (a) Cyclic
voltammograms performed with dual-mode techniques using a pair
of comb-shaped working electrodes (collector and generator) at 5 1C.
A 15 mL solution containing the DNA components (100 mM), 10 mM
phosphate buffer (pH 7.0), and 500 mM KCl was dropped on the
T
max/1C
o
Tandem duplex
with target27
DG298/
ꢀ1
ꢀ1
Lower Higher kcal mol
K
298/mol L
7
CyD-ODN
ODN7//Fc-ODN
CyD-ODN //ODN20
1
//Fc-ODN
1
37.4
19.7
18.7
24.1
73.5
73.4
73.9
73.5
ꢀ10.6
ꢀ7.36
6.52 ꢁ 10
2.50 ꢁ 10
ꢀ1
electrodes. Scan rate of generator: 10 mV s ; collector potential:
1
ꢀ
0.2 V (vs. Ag/AgCl). Solid curve in black: Fc-ODN
target27/Fc-ODN ; dotted curve: CyD-ODN /Fc-ODN
/target27/Fc-ODN . (b) Square-wave voltammograms of
/target22N/Fc-ODN . A 50 mL solution containing the
1
; broken curve:
5
1
1
1
1
; red curve:
ODN7//ODN20
CyD-ODN
CyD-ODN
1
1
a
The temperatures of biphasic UV inflections. Higher and lower
temperatures of two maxima of 1st derivative curves are shown as
2
2
DNA components (20 mM), 10 mM phosphate buffer (pH 7.0), and
b
T
maxs.z DH1 and DS1 for duplex formation of CyD-ODN
1
with half
5
00 mM KCl was subjected to measurements at 40 1C. Amplitude:
5 mV; potential increment: 4 mV; frequency: 15 Hz. Red: target22C;
duplexes are shown in ESIw. DG1 and K were calculated from them at
98 K.
2
2
blue: target22G; green: target22T; black: target22A.
This journal is c The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 12388–12390 12389

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one of the counterparts of the specific interactions of interest.
The system configuration is general and could be extended as a
common design strategy for electrochemical molecular sensing,
that is, the targets are not limited to tandem duplexes or even
DNA-based molecular systems.
This work was partially supported by a Grant-in-Aid
for Scientific Research on Innovative Areas (‘‘Coordinating
Programming’’ Area 2107, No. 22108529) from MEXT (T.I.),
Scientific Research (B) (20350038) from JSPS (T.I.).
Notes and references
Fig. 4 Switching the electrochemical signal of Fc-ODN by specific
interaction with CyD-ODN. (a) Linear calibration of the electrochemical
z Melting temperature, T
m
, is usually defined as the temperature at
response of Fc-ODN
obtained by a flow system at 25 1C. Twenty mL of standard solutions
containing various amounts of Fc-ODN , 10 mM phosphate buffer
pH 7.0), and 500 mM KCl were injected into the HPLC-ECD. To
the solution containing 100 pmol Fc-ODN , an equimolar amount of
CyD-ODN /target22C was added and its signal was measured as that of
the quenched state. Eluent: 100 mM phosphate buffer (pH 7.0), 750 mM
2
(open circle) and its quenched signal (closed circle)
which half of the ODN exists in the duplex state and the other half in
12
the single-stranded state. For the sake of convenience, here the
temperatures that give maximum of 1st derivative curves were used
as the measure for thermal stability of the duplexes.
2
(
y To determine the applied potential, hydrodynamic voltammetries
were carried out prior to conducting calibration using the same
2
3
b
2
HPLC-ECD system (ESIw).
ꢀ1
1 For example, Electrochemistry of Functional Supramolecular Systems,
ed. P. Ceroni, A. Credi and M. Venturi, Weily, Hoboken, 2010.
NaCl, 5 mg mL EDTA; potential: 500 mV (vs. Ag/AgCl);y flow
ꢀ1
rate: 1.0 mL min ; column: Inertsil AX (4.6 f ꢁ 100 mm). (b) One
2
(a) E. M. Boon, J. E. Salas and J. K. Barton, Nat. Biotechnol.,
002, 20, 282; (b) E. Farjami, L. Clima, K. Gothelf and
of the possible structures of the ternary tandem duplex (only the central
2
part of duplex 2). Red: Fc-ODN
2
, green: CyD-ODN , gray: target22C.
2
E. E. Ferapontova, Anal. Chem., 2011, 83, 1594; (c) Y. Xiao,
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GB/SA (generalized Born/surface area) solvent model using MacroModel
version 9.1.
3
The sensitivity of SWV was not sufficient. Therefore, system 2
was subjected to flow analysis to check the applicability of the
system for practical use. HPLC equipped with an electrochemical
detector (ECD) was used. Fig. 4(a) shows the calibration profile
4
5
of the electrochemical response of Fc-ODN
form. Without taking any special care, 10 pmol per 20 mL of
Fc-ODN were detected with fair reproducibility by ampero-
2
and its complex
2
metry using ordinary ECD. It would not be difficult to
improve the sensitivity by reviewing some of the parameters
of the flow system such as type of column, flow rate, and
eluent. The performance we achieved was reasonable for this
concise procedure, in which all we did was mix and inject the
components. As observed in the voltammograms of static
solutions shown in Fig. 3 and 4, the current signal was almost
perfectly suppressed by stoichiometric complexation with
1
J. Am. Chem. Soc., 2011, 133, 7676.
6 A. Kuzuya, T. Ohnishi, T. Wasano, S. Nagaoka, J. Sumaoka,
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2
0, 1643.
7
8
(a) D. R. van Staveren and N. Metzler-Nolte, Chem. Rev., 2004,
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2
9 T. Ihara and M. Mukae, Anal. Sci., 2007, 23, 625.
005, 5487.
CyD-ODN
2
in the flow system. This shows that the tandem
duplex is stable, and Fc would be steadily buried in bCyD
and shielded from the bulk solution (Fig. 4(b)), even under
the conditions of HPLC-ECD. Thus, the present system could
be applied to flow analysis without any modification of the
system architecture. Good compatibility with flow analyses
is one of the advantages of systems that work in homogeneous
10 E. Y. Krynetski, J. D. Schuetz, A. J. Galpin, C.-H. Pui,
M. V. Relling and W. E. Evans, Proc. Natl. Acad. Sci. U. S. A.,
1
995, 92, 949.
1
1 (a) D. M. Kolpashchikov, Chem. Rev., 2010, 110, 4709;
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1
2 (a) L. A. Marky and K. J. Breslauer, Biopolymers, 1987, 26, 1601;
9
solutions because, generally, flow analyses enable quick
(
b) M. Petersheim and D. H. Turner, Biochemistry, 1983, 22, 256.
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1
to applications in the micro total analyses such as mTAS or
lab-on-a-chip.
1
6
2009, 48, 2288.
1
4 (a) H. Aoki, A. Kitajima and H. Tao, Supramol. Chem., 2010,
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In conclusion, we have shown that the electrochemical
activity of Fc is controlled reversibly by a designed interaction
with bCyD on a DNA scaffold. bCyD could be regarded as an
effective quencher for Fc. This would free the design of the
molecular sensor from the need for electrode modification of
1
1
5 For example, M. Zuker, Nucleic Acids Res., 2003, 31, 3406.
6 For example, Nanobiotechnology, Concepts, Applications and
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1
2390 Chem. Commun., 2011, 47, 12388–12390
This journal is c The Royal Society of Chemistry 2011