Characterization of PEDOT film functionalized with a series of automated synthesis ferrocenyl-containing oligonucleotides

Article abstract of DOI:10.1016/j.tet.2005.02.058

In previous works we have described a fully automated synthesis of new ferrocene labelled oligonucleotides (Fc-ODNs) probes with one or more electroactive markers at different position in the chain. These Fc-ODNs have shown good properties to detect ODN target in solution. Here we describe the post-functionalization of a conducting co-polymer based on ethylenedioxythiophene (EDOT) derivatives by a series of Fc-ODNs. The grafting of the Fc-ODNs probes resulted in the appearance of the ferrocene redox couple which directly confirm the effectiveness of the ODN anchoring compared to traditional approach based on IR spectroscopy and X-ray fluorescence of the films. Moreover, the electrochemical response of the modified electrodes analysed in organic media before and after hybridization with ODN target confirm that properties obtain in solution for Fc-ODNs already exist in the film. The changes in the current intensity were found to be dependant on the structure of the grafted ODN that validate our strategy to synthesize an optimal Fc-ODNs.

Full text of DOI:10.1016/j.tet.2005.02.058

Tetrahedron 61 (2005) 3947–3952
Characterization of PEDOT film functionalized with a series of
automated synthesis ferrocenyl-containing oligonucleotides
a
a
a
a,
*
´ ´
Aude-Emmanuelle Navarro, Frederic Fages, Corinne Moustrou, Hugues Brisset,
Nicolas Spinelli,b Carole Chaixb and Bernard Mandrandb
a
´ ´ ´
Groupe de Chimie Organique Materiaux Moleculaires, UMR CNRS 6114, Faculte des Sciences de Luminy, 163 avenue de Luminy,
13288 Marseille Cedex 9, France
Systemes Macromoleculaires et Immunovirologie Humaine, UMR CNRS 2714, Ecole Normale Superieure de Lyon, 46 allee d’Italie,
69007 Lyon, France
b
`
´
´
´
Received 15 November 2004; revised 21 February 2005; accepted 22 February 2005
Abstract—In previous works we have described a fully automated synthesis of new ferrocene labelled oligonucleotides (Fc-ODNs) probes
with one or more electroactive markers at different position in the chain. These Fc-ODNs have shown good properties to detect ODN target in
solution. Here we describe the post-functionalization of a conducting co-polymer based on ethylenedioxythiophene (EDOT) derivatives by a
series of Fc-ODNs. The grafting of the Fc-ODNs probes resulted in the appearance of the ferrocene redox couple which directly confirm the
effectiveness of the ODN anchoring compared to traditional approach based on IR spectroscopy and X-ray fluorescence of the films.
Moreover, the electrochemical response of the modified electrodes analysed in organic media before and after hybridization with ODN target
confirm that properties obtain in solution for Fc-ODNs already exist in the film. The changes in the current intensity were found to be
dependant on the structure of the grafted ODN that validate our strategy to synthesize an optimal Fc-ODNs.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
films are generally necessary to confirm the grafting of
ODNs19 and no quantitative determination of the amount of
ODN immobilized on the polymer film could be achieved.
Moreover, the common functional feature pertaining to
these systems is the intensity decrease of the electro-
chemical signal in response to hybridization, which is
detrimental to the sensitivity performance of the sensor.
The development of efficient methods for real-time
detection of specific DNA sequences is currently receiving
a tremendous amount of interest for applications in clinical
diagnostics1 environmental protection,2,3 food quality
control,4 and forensic science.5 In this context electro-
chemical sensor devices have emerged as promising tools
due to their high sensitivity, easy implementation, low
production cost and ability to miniaturization.6–8 Such
systems are based on the covalent immobilization of probe
oligonucleotides (ODNs) onto a surface, which allows the
direct detection of the hybridization reaction with the target
ODN.9 In that connection, conjugated polymers as poly-
thiophene or polypyrrole did appear as valuable conducting
substrates for the grafting of the ODN probes. Indeed the
change in either the intrinsic electroactivity of the conduct-
ing backbone or the electrochemical properties of a pending
redox label can be used as a transduction mechanism to
monitor the hybridization reaction with the target ODN.9–18
However, IR spectroscopy and X-ray fluorescence of the
In this context, we have developed a new strategy based on
the post-functionalization of a conducting polymers by
ODN probes and ferrocene units at the same place and in a
one-pot reaction. Indeed the ferrocene moiety displays a
reversible and narrow redox behavior that is sensitive to
electronic and steric factors. By this way the appearance of
the ferrocene redox couple in cyclic voltammetry (CV)
directly confirm the effectiveness of the ODN anchoring
and the exact integration of the amount of charge
exchanged by ferrocenyl groups allows to the electrode
coverage.
In previous works we have prepared and demonstrated the
good capacity to detect ODN target of a series of ODNs
bearing different number of ferrocene unit directly
incorporated into the base sequences during the automated
solid phase (Chart 1).20–22
Keywords: Modified electrodes; Ethylenedioxythiophene; Electrochemical
biosensing; Ferrocene.
Corresponding author. Tel.: C33 4 91 82 95 87; fax: C33 4 91 82 95 80;
e-mail: brisset@luminy.univ-mrs.fr
*
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.02.058

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A.-E. Navarro et al. / Tetrahedron 61 (2005) 3947–3952
Chart 1. List of ferrocene Fc-ODNs used for film preparation.
In this paper, the post-functionalization of a poly(ethylene-
dioxythiophene) (PEDOT) film with the Fc-ODN systems
was performed. So far the PEDOT backbone has not been
investigated for the generation of DNA sensors although it
represents a preferential conducting polymer for such
application owing to its high chemical stability, compati-
bility with aqueous media, and conductivity.23 According to
our previous results on PEDOT-based modified
electrodes,24 we describe here the synthesis of a novel
hydroxysuccinimidyl ester derivative of EDOT, 1, and the
electrochemical copolymerization of 1 and the bis-EDOT
compound, 2 (Scheme 1).25 It is shown that a redox signal
corresponding to the ferrocene units appear upon covalent
immobilization of ferrocene-ODN assemblies via amide
bond formation, which directly confirm, the probes grafting.
62.5 MHz, respectively. UV spectra were obtained on a
Varian Cary 1E spectrophotometer. Mass spectrometry was
performed with a JEOL FX 102 Mass spectrometer in the
FAB mode (Laboratoire de Mesures Physiques, USTL,
Montpellier, France). MALDI-TOF mass spectra of oligo-
nucleotides were recorded at the IBCP, Lyon on a Voyager
DE (Perseptive Biosystems, Framingham, MA, USA) mass
spectrometer equipped with an N2 Laser. The matrix used
for oligonucleotide mass analyses was hydroxypicolinic
acid (HPA). Calibration was carried out using internal data
base reference.
2.3. Synthesis of functionalized EDOT
2.3.1. 3-(2,3-Dihydro-thieno[3,4-b][1,4]dioxin-2-yl-
methoxy)-propionic acid ethyl ester 3. A mixture of
NaH (110 mg, 2.73 mmol, 60% in mineral oil) and 18-
crown-6 ether (13 mg, 0.05 mmol) in 2.8 mL of anhydrous
THF was stirred under argon at K10 8C. 2,3-Di-
hydrothieno[3,4-b][1,4]dioxin-2-yl)methanol (Hydroxy-
methyl EDOT) (280 mg, 1.63 mmol) in 2.8 mL of
anhydrous THF was added. The mixture was stirred at
room temperature for 1 h and then cooled down to 0 8C.
Ethyl-3-bromopropionate (500 mg, 2.76 mmol) in 2.8 mL
of anhydrous THF was added drop by drop during 10 min.
After 72 h under stirring at room temperature the mixture
was diluted with 25 mL of 5% NH4Cl aqueous solution. The
mixture was extracted with diethylether (3!25 mL). The
combined organic phases were washed with water, dried
over MgSO4, and evaporated in vacuo. The crude product
was purified on a silica gel column using cyclohexane–ethyl
acetate (70:30 v/v) as eluent to afford 4 (93 mg, 21%) as a
Scheme 1. Precursors of copolymer.
2. Experimental
1
pale yellow oil. H NMR (270 MHz, CDCl3) d: 1.12 (dd,
3JZ7.2 Hz and 3JZ7.0 Hz, 3H, CH3), 2.44 (dd, 3JZ6.5 Hz
2.1. Reagents
3
and JZ6.2 Hz, 2H, CH2–CO), 3.46–3.59 (m, 2H, CH2O),
3.64 (t, 3JZ6.2 Hz, 2H, OCH2 CH2CO), 3.84–4.19 (m, 5H,
CH3–CH2O, CH, CH2–CH), 6.17 (s, 2H, Hthiophene) ppm.
13C NMR (67.5 MHz, CDCl3) d: 14.2, 35.0, 60.6, 66.0, 67.2,
69.3, 74.5, 99.7, 141.5 (CH), 171.3 (C]O). MS (FAB): 273
(MC1, 16), 272 (M%C, 12), 89 (16), 55 (11), 29 (2).
Lithium hydride (LiH), 18-crown-6, ethyl-3-bromo-
propionate, 1-(3-dimethylaminopropyl)-3-ethylcarbo-
diimide hydrochloride, N-hydroxysuccinimide were
purchased from Aldrich and used as received. THF
(anhydrous) and DMF (anhydrous) were purchased from
ACROS. Diethylether, absolute ethanol, ethyl acetate,
cyclohexane were purchased from CarloErba, silica gel
(240–400 mesh) from Merck and MgSO4 from SDS. All
aqueous solutions were made with MilliQ purified water
(Maxima ELGA). Phosphate buffers (pHZ6.8) were made
with 0.25 M KH2PO4, 0.25 M Na2HPO4 and 0.75 M NaCl.
2,3-Dihydrothieno[3,4-b][1,4]dioxin-2-yl)methanol
(hydoxymethyl-EDOT) and hexaethylene glycol Bis(2,3-
dihydrothieno[3,4-b][1,4]-dioxin-2-ylmethyl)ether 2 were
prepared as described in the literature.23,25
2.3.2. 3-(2,3-Dihydro-thieno[3,4-b][1,4]dioxin-2-yl-
methoxy)-propionic acid 4. Compound 3 (37 mg,
0.14 mmol) in 2 mL of absolute ethanol and 2 mL of an
aqueous solution of NaOH (22 mg, 0.54 mmol) were stirred
at reflux during 6 h. After the reaction mixture was cooled to
room temperature, the solvents were evaporated in vacuo.
The crude product was dissolved in 40 mL of water and
extracted with diethylether (2!30 mL). The aqueous phase
was acidified with HCl 2 M until acidic pH and extracted
again with diethylether (2!30 mL). The second combined
organic layers were dried over MgSO4, and evaporated in
vacuum to afford 33 mg (0.14 mmol, quantitative yield) of a
white powder corresponding to compound 4: mp 76–78 8C;
2.2. Physical and spectroscopic methods
Melting point are uncorrected and were measured on a
1
3
Electrothermal 9100 apparatus. H and 13C NMR spectra
were recorded on a Bruker AC 250 spectrometer at 250 and
1H NMR (270 MHz, C5D5N) d: 4.08 (dd, JZ6.0 Hz and
3JZ6.6 Hz, CH2COOH), 4.93–5.07 (m, 2H, CHCH2O),

A.-E. Navarro et al. / Tetrahedron 61 (2005) 3947–3952
3949
3
5.19 (t, JZ6.3 Hz, 2H, OCH2CH2COOH), 5.35–5.63 (m,
with 1 mL of CH3CN/H2O (1:1) and solvents were
evaporated to dryness. Then, ODN was detritylated with
300 mL of 80% acetic acid/water during 30 min. Acetic acid
was eliminated by evaporation and ODN was precipitated
twice with 0.3 M AcONa (300 mL) and ethanol (900 mL)
before lyophilization in water. Oligonucleotide was purified
by HPLC on a RP-18e Lichrospher 100 (300!7.5, 10 m,
Merck) with linear gradient of acetonitrile (5–40%) in
0.05 M aqueous triethylammonium acetate (pHZ7). HPLC
analyses of ODN purities was run on a reverse-phase RP-
18e Chromolith Performance column (100!4.6 mm,
Merck) with the same eluents than above described. ODN
3.3.3 was characterized by MALDI-TOF mass spectral
analyses. The result (m/z calcd 8039.05, found 8042,40.70)
illustrate the successful incorporation of ferrocenyl moieties
into the oligonucleotide. The difference between calculated
and found masses is attributed to the calibration of the
instrument with standards with a behavior similar to
ferrocenyl-ODN. The same problem was observed in
literature.22,26
2H, CH2CH), 5.64–5.66 (m, 1H, CH), 7.86 (s, 2H,
Hthiophene), 10.0 (s, 1H, OH) ppm. 13C NMR (62.5 MHz,
C5D5N) d: 37.8, 68.4, 70.0, 71.6, 75.2, 102.2 (CH), 175.9
(C]O) ppm. MS (FAB): 244 (M%C, 50); 89 (21). Anal.
Calcd: C, 49.17; H, 4.95. Found: C, 49.33; H, 4.89.
2.3.3. 3-(2,3-Dihydro-thieno[3,4-b][1,4]dioxin-2-yl-
methoxy)-propionic acid 2,5-dioxo-pyrrolidin-1-yl ester
1. A mixture of 4 (110 mg, 0.45 mmol), 1-(3-dimethyl-
aminopropyl)-3-ethylcarbodiimide hydrochloride (121 mg,
0.63 mmol), N-hydroxysuccinimide (62 mg, 0.54 mmol) in
2 mL of anhydrous DMF was stirred under argon atmo-
sphere at room temperature during 20 h. After addition of
acetone (10 mL) and water (10 mL), the mixture was
extracted (3!30 mL) with a mixture of diethylether and
ethylacetate (2/1 v/v). The combined organic layers were
washed with water, dried over MgSO4, and evaporated in
vacuum to afford 154 mg (0.45 mmol, quantitative yield) of
1 as a white solid: mp 84–85 8C; 1H NMR (CDCl3) d: 2.63
3
3
(s, 4H, CH2 (succi)), 2.70 (dd, JZ6.0 Hz and JZ6.3 Hz,
2H, CH2CO), 3.52 (m, 2H, CH2O), 3.69 (t, 3JZ6.0 Hz, 2H,
OCH2CH2CO), 3.84–4.08 (m, 2H, CH2CH), 4.10–4.15 (m,
1H, CH), 6.13 (s, 2H, CHthiophene) ppm. 13C NMR (CDCl3)
d: 26.4, 33.0, 66.8, 67.1, 70.3, 73.2, 100.4, 142.4, 167.4
(C]O), 169.8 (C]O) ppm. MS (FAB): 341 (M%C, 10).
HRMS (FAB): calcd for C14H15O7NS 341.0569, found
341.0564. Anal Calcd: C, 49.26; H, 4.43; O, 32.81; N, 4.10;
S, 9.39. Found: C, 50.08; H, 4.98; O, 29.20; N, 3.83; S, 8.08.
2.5. Electrochemical materials
Cyclic voltammetry data were acquired using a computer-
based Bioanalytical instrument (BAS 100) electrochemical
workstation with a three-electrode setup. A 1.6 mm
diameter gold working electrode was used and polished
with 1–0.1 mm diamond paste and ultrasonically rinsed in
absolute ethanol. Counter electrode in platinum and
Ag/AgCl (with 3 M NaCl filling solution) reference
electrode were used. All anhydrous solvent are of electronic
grade purity.
2.4. Ferrocenyl-labelled oligonucleotides
Ferrocenyl-labelled oligonucleotides ODN 5.3 and 3.3 were
synthesized as previously described.20–22 The ODN probes
have a terminal amino group at the 30 position allowing
covalent linking on the copolymer film (Chart 1). Oligo-
nucleotide target, complementary to the oligonucleotide
probes with 50-ACC-TTA-TGA-GTC-CAA-GGA-ATA-C-
30 sequence was provided by the bioMerieux company. The
noncomplementary oligonucleotide is T20: 30-TTT-TTT-
TTT-TTT-TTT-TTT-T-50.
2.6. Electrochemical polymerization
The copolymer was obtained from electropolymerization of
precursor 1 and 2 in acetonitrile containing 10K1
M
nBu4NCF3SO3 as supporting electrolyte at a fixed potential
of 1.3 V/Ag/AgCl. Therefore, their initial concentration
were kept the same: 0.01 M. 5 mC cmK2 were deposited.
Acetonitrile with 10K1 M nBu4NCF3SO3 was used to
analyse the co-polymers. Cyclic voltammograms were
recorded without ohmic drop compensation and at a scan
rate of 100 mV/s. All cyclic voltammetry experiments were
carried out at 25 8C using a cell equipped with a jacket
allowing circulation of water from the thermostat.
ODN 3.3.3 containing free ferrocenyl groups was syn-
thesized using an Applied Biosystems 394 RNA/DNA
synthesizer. 1-[3-O-dimethoxytritylpropyl]-10-[30-O-(2-
cyanoethyl-N,N-diisopropyl phosphoramidityl) propyl]fer-
rocene20 was dissolved in anhydrous acetonitrile (CZ
˚
0.09 M) and the solution was dried on 3 A molecular sieves
during 24 h. Solution was filtered on 0.22 mm PVDF filter
before loading on DNA synthesizer. Standard 1 mmol
coupling cycle was used for oligonucleotide elongation.
The coupling reaction time was increased from 15 to 500 s
for ferrocene synthons. After ODN synthesis (DMTr ON),
controlled pore glass (CPG) support was treated in NH4OH
(30% aqueous) during 16 h at 55 8C, then supernatant was
recovered and evaporated to dryness under vacuum. The
pellet was dissolved in 500 mL of MilliQ water plus 500 mL
of MOP buffer. ODN was purified on MOP column (CTGen,
San Jose, CA) as followed: column was first washed with
2 mL of CH3CN/H2O (1:1) and 2 mL of TEAAc buffer
0.1 M (pHZ7) successively before loading the ODN
solution on column. Truncated sequences were eluted with
4 mL of TEAAc buffer 0.1 M (pHZ7). ODN was eluted
2.7. Covalent immobilization of the ferrocene-ODN
probes onto PEDOT
Substitution of the succinimidyl ester groups of the
copolymer using the amino Fc-ODN probes was carried
out by simple immersion of the modified electrode during
3 h at 25 8C in acetonitrile/phosphate buffer pH 6.8
(80/20 v/v) solution of the Fc-ODN probe (6.25 mM). To
remove any ungrafted Fc-ODN probes, the modified
electrode was thoroughly washed with water. The grafted
modified electrode was then analysed by cyclic voltam-
metry in acetonitrile solution containing 10K1 M nBu4-
NCF3SO3 as supporting electrolyte.

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A.-E. Navarro et al. / Tetrahedron 61 (2005) 3947–3952
2.8. Hybridization of ODN target
3.2. Co-electropolymerization of precursors
PEDOT-ODN 5.3, 3.3 and 3.3.3 modified electrodes were
incubated at 37 8C during 3 h in a phosphate buffer (pH 7.4)
containing the ODN target (6.25 mM). After incubation, the
modified electrodes were rinsed with the same buffer and
electrochemically characterized in acetonitrile solution
containing 10K1 M nBu4NCF3SO3 as supporting
electrolyte.
The precursor copolymer was formed easily on the gold
electrode by electropolymerization of monomers 1 and 2
(10K2 M each) in an acetonitrile solution of nBu4NCF3SO3
(10K1 M). Monomers 1 and 2 were shown to polymerize at
the same oxidation potential.24 The 5 mC cmK2 film was
grown in potentiostatic conditions at 1.3 V/Ag/AgCl. The
peak current of the electrochemical response of copolymer
in acetonitrile was found to increase linearly with the scan
rate, as expected for a confined surface electrochemical
process with the formation of a stable adhesive film (Fig. 1).
The cyclic voltammogram (CV) of the resulting co-polymer
shows two anodic waves around K0.16 and C0.25 V
characteristic of the PEDOT backbone. In order to test the
influence of different solutions on the co-polymer, it was
immersed 3 h at 25 8C in acetonitrile/phosphate buffer
solution without ODN probes then washed and immersed
3 h at 37 8C in phosphate buffer without ODN target. The
CV before and after immersion are similar. A little decrease
of the current intensity is observed for the two redox
systems of the PEDOT after the second immersion with a
40 mV negative shift for the first oxidation peak. This
behavior reflects the change in solvation of the co-polymer
3. Results and discussion
3.1. Synthesis of the functionalized EDOT monomer 1
The synthetic route toward 1 is outlined in Scheme 2. The
ester 3 was synthesized in 21% yield from hydroxymethyl
EDOT by a Williamson reaction with ethyl-3-bromo-
propionate. The hydrolysis of ester group was performed
in presence of NaOH to give quantitatively the carboxylic
acid 4. Reaction of 4 with N-hydroxysuccinimide in the
presence of 1-(3-dimethylaminopropyl)-3-ethylcarbo-
diimide hydrochloride reagent lead quantitatively to the
precursor 1.
Scheme 2. Synthesis of precursor 1.
when exposed to different solutions. Repetitive scans did
not induce any change in the CV curves, which indicated a
good stability of the PEDOT film.
3.3. Covalent immobilization of the ferrocene-ODN
probes
After covalent grafting of the Fc-ODN probes, a small
decrease of the current intensity of the first redox system of
the copolymer was observed and attributed to the solution
effects (Fig. 2 and Table 1). In contrast no decrease of the
current intensity was observed for the second redox system
of the copolymer except for ODN 3.3.3. In all cases a new
redox system corresponding to the oxidation and subsequent
reduction of ferrocenyl group was observed. The current
intensity of the ferrocene redox system was observed to be
depending on the nature of the ODN probe.
Figure 1. Cyclic voltammogram of copolymer in 0.1 M nBu4NCF3SO3/
CH3CN, scan rate 100 mV sK1. (Film grown under potentiostatic
conditions, see text). Before immersion in solutions (dotted line); after
immersion in grafting solution (dashed line); after immersion in grafting
and hybridization solutions (solid line).
With ODN 5.3 grafted onto co-polymer the anodic shoulder

A.-E. Navarro et al. / Tetrahedron 61 (2005) 3947–3952
3951
the oxidation and reduction peaks being respectively
observed at 0.33 and 0.32 V.
The use of Fc-ODN probes allow direct assessment of the
linkage of the ODN probes on the polymer film by
visualization of the ferrocenyl electrochemical response
that increases with the number of ferrocene units. Moreover,
by exact integration of the areas under the ferrocene anodic
peaks corrected from background current the efficiency of
the post-functionalization of the co-polymer can be
estimated. The exact integration of the amount of charge
exchanged by ferrocenyl groups during the CV for PEDOT-
ODN 3.3.3 corrected from background current corre-
sponded to an electrode coverage of 78 pmol cmK2
.
3.4. Incubation with non-complementary DNA strand
The PEDOT-Fc-ODN modified electrode were incubated at
37 8C during 3 h in a phosphate buffer (pH 7.4) containing
the non-complementary ODN target (6.25 mM). After
incubation, the modified electrode was rinsed with the
same buffer solution and electrochemically characterized in
acetonitrile. In all cases the electrochemical response of the
modified electrodes before and after incubation with non-
complementary ODN target were identical (Fig. 3).
3.5. Hybridization with ODN target
The hybridization of PEDOT-ODN 5.3 and 3.3 with the
complementary ODN target led to an increase of the current
intensity and a 60 mV negative shift for all oxidation waves.
The oxidation peaks of the ferrocenyl group shift from 0.35
to 0.29 and from 0.36 to 0.30 V for PEDOT-ODN 5.3 and
3.3, respectively. The negative shift of potentials can be
attributed to a faster electron transfer through the copolymer
after hybridization. Pharm et al. interpreted the current
enhancement upon hybridization on the basis of changes in
the conformation of the ODN strands. Indeed, single-
stranded ODNs behave as random coils while after
hybridization of a complementary sequence the double-
stranded ODNs lead to a more organized surface, through
which counter ions could diffuse more freely.9 Additionally
it is believed that the hydrophilic character of hybridization
of DNA combined to the highly hydrophilic character of
EDOT-based polymers containing oligooxyethylene chains
can increase the permeability of the polymer films to doping
anions. With PEDOT-ODN 3.3.3 the hybridization induces
a decrease in the current density together with a positive
shift of the oxidation wave, as generally observed with
conjugated polyheterocycles functionalized with ODN
recognition centres. The decrease of the current intensity
and a positive shift of oxidation potential peak were
explained by a decrease of the permeability of the polymer
Figure 2. Cyclic voltammograms between K400 and 500 mV of
functionalized copolymers in 0.1 M nBu4NCF3SO3/CH3CN, scan rate
100 mV sK1. Before grafting (dotted line) and after grafting with a Fc-ODN
(dashed line); after hybridization with target ODN (solid line).
associated with the ferrocene system was observed at ca.
0.3–0.4 V. The CV of co-polymers grafted with ODN 3.3
show that the anodic shoulder associated with the ferrocene
system become an anodic wave accompanied by the
emergence of the subsequent cathodic shoulder. The most
important effect is obtained with PEDOT-ODN 3.3.3. The
CV curve of PEDOT-ODN 3.3.3 exhibit the characteristic
peaks of ferrocenyl groups with a quasi-ideal redox system,
Table 1. Electrochemical values for the grafting of ODN-Fc and hybridization with complementary DNA target
ODN entry
Probe surface density corrected
from background (pmol cmK2
Variation of peak height of ferrocene oxidation wave after hybridization
corrected from background grafting curve (mA)
)
ODN 5.3
ODN 3.3
ODN 3.3.3
32 (12)
46 (10)
78 (8)
0.37 (0.05)
0.27 (0.05)
K0.10 (0.03)
The values in parentheses represent the standard deviations from several electrode measurements (nO4) not the nonlinear curve fitting errors.

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A.-E. Navarro et al. / Tetrahedron 61 (2005) 3947–3952
Acknowledgements
The authors wish to thank Dr. J. Roncali from University of
Angers for helpful discussions. The authors thank Linda
Danao for checking the manuscript.
References and notes
1. MacPherson, J. M.; Gajadhar, A. A. Mol. Cell. Probes 1993, 7,
97.
Figure 3. Cyclic voltammogram of PEDOT-ODN 3.3.3 between K400
.
Before grafting (dotted line), after grafting Fc-ODN (dashed line) and after
incubation with non-complementary ODN target (circle dashed line).
2. Mathe, J.; Eisenmann, C.; Seitz, A. In DNA Fingerprinting:
State of the Science; Pena, S. D. J., Chakraborty, R., Epplen,
and 600 mV in 0.1 M nBu4NCF3SO3/CH3CN, scan rate 100 mV sK1
¨
T. J., Jeffrey, A. J., Eds.; Birkhauser: Basel, 1993; pp 387–393.
3. Lucarelli, F.; Palchetti, I.; Marrazza, G.; Mascini, M. Talanta
2002, 56, 949.
films to doping anions and to the change of conformation of
the conjugated backbone.11–16
4. Carnegie, P. R. Australas. Biotechnol. 1994, 4, 146.
5. Rao-Coticone, S.; Collins, P.; Dimsoski, P.; Ganong, C.;
Hennessy, L.; Leibelt, C.; Shadravan, F.; Reeder, D. Int.
Congress Ser. 2003, 3.
These results are very interesting for the development of
direct DNA hybridization electrochemical sensors as, to our
knowledge, there is no reported example in the literature
describing an increase of the current intensity after ODN
probes linkage followed by a new enhancement of current
intensity after hybridization of the ODN target. Moreover,
this strategy allows the determination of the optimal
position and the number of the ferrocenyl groups into the
ODN probe sequences.
6. Mikkelsen, S. R. Electroanalysis 1996, 8, 15.
7. Wang, J.; Cai, X.; Rivas, G.; Shiraishi, H.; Dontha, N. Biosens.
Bioelectron. 1997, 12, 587.
8. Drummond, T. G.; Hill, M. G.; Barton, J. K. Nature
Biotechnol. 2003, 21, 1192.
9. Pham, M. C.; Piro, B.; Tran, L. D.; Ledoan, T.; Dao, L. H.
Anal. Chem. 2003, 75, 6748 and references therein.
10. Wang, J.; Jiang, M. Langmuir 2000, 16, 2269.
11. Korri-Youssoufi, H.; Garnier, F.; Srivastava, P.; Godillot, P.;
Yassar, A. J. Am. Chem. Soc. 1997, 119, 7388.
4. Conclusion
¨
12. Bauerle, P.; Emge, A. Adv. Mater. 1998, 3, 324.
¨
13. Emge, A.; Bauerle, P. Synth. Met. 1997, 84, 213.
A succinimidyl ester derivative of EDOT has been
synthesized and characterized. Its co-electropolymerization
with oligo(oxyethylene)diEDOT in potentiostatic con-
ditions in acetonitrile leads to a stable film. The use of
this new copolymer film for the immobilization of
ferrocene-modified ODN probes shows a sensitive and
selective DNA sensor. Indeed hybridization of ODN target
is observed to induce a positive shift of the oxidation waves
and an increase of the current intensity. The latter increase is
more important when three ferrocenyl groups are near the
electrode surface. These results shows great promise for the
development of direct DNA hybridization electrochemical
sensors, as to the best of our knowledge, there are so far no
examples reported in literature which describes an increase
of the current intensity after ODN probes immobilization
followed by a new enhancement of current intensity after
hybridization of the ODN target. Moreover, this work also
emphasizes the subtle balance between the number and
position of the ferrocenyl groups in ODN probe sequence in
order to achieve accurate electrochemical sensing of DNA
hybridization.
14. Cha, J.; Im Han, J.; Choi, Y.; Sung Yoon, D.; Oh, K. W.; Lim,
G. Biosens. Bioelectron. 2003, 18, 1241.
15. Korri-Youssoufi, H.; Makrouf, B. Anal. Chim. Acta 2002, 469,
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16. Makrouf, B. Thesis, University of Paris 7, 2002.
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F.; Mandrand, B.; Spinelli, N.; French, N. Patent 2002, FR
02/01858; PCT WO 03/068787 A1.
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Lastly this works validate ours simple strategy to prepare
labelled ODN with one or more electroactive markers at
different positions of the chain22 because it is necessary to
test different combination of Fc-ODNs to obtain the best
DNA sensors. Work to prepare ODN probes with two and
three ferrocenyl groups in 30 position with one and more 50
ferrocenyl groups is currently underway.
´
25. Perepichka, I. F.; Besbes, M.; Levillain, E.; Salle, M.; Roncali,
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