Synthesis and characterization of novel conducting homopolymers based on amino β-styryl terthiophene

Article abstract of DOI:10.1139/V08-136

Novel ECPs (electronic conducting polymers) based on amino β-styryl-substituted terthiophene (AST) were synthetized by direct electropolymerization. The ECPs were characterized by cyclic voltammetry, square-wave voltammetry, Fourier transform infrared (FT

Full text of DOI:10.1139/V08-136

1010
Synthesis and characterization of novel
conducting homopolymers based on amino
␤-styryl terthiophene
Hakim Mehenni and Lê H. Dao
Abstract: Novel ECPs (electronic conducting polymers) based on amino β-styryl-substituted terthiophene (AST) were
synthetized by direct electropolymerization. The ECPs were characterized by cyclic voltammetry, square-wave
voltammetry, Fourier transform infrared (FTIR) spectroscopy, and X-ray photoelectron spectroscopy (XPS). The
poly(amino β-styryl terthiophene) displayed cyclic and square-wave voltammograms with redox peaks that can be as-
signed to the aminophenyl moiety and the polyterthiophene backbone. The presence of free primary amine groups on
the ECP film permitted further biological functionalization (i.e., covalent bonding of various bioreceptors on its sur-
face). The electrochemical performance of Biotin grafted at the AST modified glassy carbon electrode was investigated
to detect the Avidin protein in solution by cyclic voltammetry and square-wave voltammetry.
Key words: electronic conducting polymer, electrode surface modification, biosensor, β-styryl-substitued terthiophene,
functionalization, cyclic, square-wave voltammetry.
Résumé : Faisant appel à une électropolymérisation directe, on a réalisé la synthèse de nouveaux polymères conduc-
teurs électroniques (PCE) à base de terthiophènes portant des substituants β-styryles aminés (TSA). On a caractérisé les
PCE par voltampérométrie cyclique, par voltampérométrie à onde carrée, par spectroscopie infrarouge à transformée de
Fourier (IR-TF) et par spectroscopie photoélectronique de rayons X (SPX). Les poly(amino β-styryl terthiophènes) pré-
sentent des voltampérogrammes cyclique et à onde carrée comportant des pics redox qui peuvent être attribués à la
portion aminophényle ainsi qu’au squelette polyterthiophène. La présence de groupes amines primaires libres sur le
film de PCE a permis de réaliser une autre fonctionnalisation biologique, c’est-à-dire la formation de liaisons covalen-
tes de divers biorécepteurs sur sa surface. On a évalué la performance électrochimique de la biotine fixée sur une élec-
trode de carbone vitreuse modifiée par des TSA à détecter la protéine avidine en solution par les techniques de
voltampérométrie cyclique et à onde carrée.
Mots-clés : polymère conducteur électronique, modification de la surface d’une électrode, biosenseur, terthiophène
portant une β-styryle substitué, fonctionnalisation, voltampérométrie cyclique et à onde carrée.
[Traduit par la Rédaction] Mehenni and Dao 1018
Introduction
prepared by immobilizing the molecular recognition
bioreceptor onto the substrate materials (e.g., ECP), and this
bioreceptor (e.g., antigen, biotin) recognizes a biomolecule
compound (e.g., antibody, avidin) according to its specific-
ity. Therefore, bioreceptors have to be properly immobilized
on the sensor surface, and a transducer signal has to be gen-
erated, which is related to the concentration of the
biomolecule in the sample (6). This kind of device is called
reagentless “third-generation biosensor” in which a redox
probe is not required (7).
In recent years, conducting polymers have been the sub-
ject of considerable academic and industrial research be-
cause of their potential uses in areas such as storage and
energy conversion, electrocatalysis, corrosion-resistant mate-
rials, and sensors (1–3). Moreover, the use of electronic con-
ducting polymers (ECP) as an immobilization matrix of
biomolecules on the electrode surface and as an electronic
transducer has taken a considerably important place in the
development of biosensors (4, 5). Generally, a biosensor,
consisting of a transduction system coupled to a receptor, is
Oligo and polythiophenes have also been functionalized
as advanced materials for electrode surface modifications
allowing the development of chemosensor (8–10) and bio-
sensor devices (11–13). Effectively, the advantage of making
polythiophene films from functionalized terthiophene is to
avoid reaching a highly positive potential, which may cause
the degradation of delicate functional groups because
terthiophene has a lower oxidation potential than thiophene
or bithiophene; thus, electrochemical polymerization is eas-
ier (14). Moreover, conducting polymers from terthiophene
Received 12 March 2008. Accepted 23 July 2008. Published
on the NRC Research Press Web site at canjchem.nrc.ca on
3 October 2008.
H. Mehenni and L.H. Dao1. Laboratoires de Recherche sur
les Matériaux Avancés, INRS-Energie Matériaux
Télécommunications, 1650 Bd Lionel Boulet, Varennes, QC
J3X 1S2, Canada.
1Corresponding author (e-mail: le.dao@emt.inrs.ca).
Can. J. Chem. 86: 1010–1018 (2008)
doi:10.1139/V08-136
© 2008 NRC Canada

Mehenni and Dao
1011
are known for their simple functionalization and relatively
good stability in air for the neutral and oxidized states (15).
Reeman et al. (16) developed an avidin biosensor based
on a direct functionalization of terthiophene by biotin.
Biotin was directly bonded to the central thiophene ring in
the 3′ position by chemistry synthesis. A redox-inactive
polythiophene film was obtained from an electrochemical
polymerization of the biotin-functionalized terthiophene
alone, because the bonded biotin has an insulating effect on
the polythiophene backbone. Consequently, a polythiophene
copolymer was realized from the biotin-functionalized
visible to low potential (towards 0.4 V vs. Ag/AgCl), and
were able to be exploited by a simple electrochemical tech-
nique such as cyclic voltametry to detect and to measure
cytochrome C. On the other hand, the disadvantage of this
biosensor system based on the poly(terthiophene 3′-
ferrocene) film is that its use is restricted to a single biologi-
cal target, here Cytochrome C. Also, the ferrocene ring is
known for its toxicity in situ to the point that some of these
by-products are the object of interest as anticancerous agents
(21–23). This constitutes a major disadvantage in the devel-
opment of biosensors for an in situ application in a medical
field.
′′
terthiophene and 2,2′:5′,2 -terthiophene (1:5 molar ratio, re-
To bring a solution to the problematic context above, we
chose to combine the strategies of Shim and Wallace. Herein,
we report the synthesis and the characterization of novel
conducting homopolymers based on amino β-styryl terthio-
phene, which have never been reported. The amino β-styryl
terthiophene monomer corresponds to the functionalization
of the terthiophene by an amino β-styryl group, covalently
bonded to the central thiophene ring in the 3′ position.
We have synthetized the 3′-(3-methyl-4-amino-β-styryl)-
spectively) to obtain a redox-active copolymer film. Another
inconvenience was that the polythiohene redox signal was
situated at a high potential, approximately 1 V vs. Ag/AgCl,
leading usually to the degradation of the bioreceptor, i.e., of
biotin. Also, no redox signal was clearly visible to allow the
measurement of avidin by using a simple electrochemical
method as cyclic voltammetry or square-wave voltammetry;
for this reason, impedance was used without forgetting the
inconvenience of using chemical synthesis to make biologi-
cal functionalization of polythiophene film, because this
type of biosensor system will be limited only for a single
target biomolecule.
A new approach to the functionalization of the surface of
polythiophene films appeared with the work of Shim et al.
The terthiophene was functionalized by a carboxylic acid
group (–COOH) in the 3′ position of the central thiophene
ring to prepare the terthiophene 3′-carboxylic acid (TCA).
Biosensors of vitellogenin (17), glutamatate (18), and H2O2
(19) have been developed after the biological surface
functionalization of the poly(TCA) film electrodeposited on
an electrode surface. The advantage of biosensor systems
based on the poly(TCA) films is to be not restricted to a sin-
gle target biomolecule. Indeed, various bioreceptors can be
immobilized through a covalent bond by interaction with
free carboxylic acid groups present on the poly(TCA) film
surface. The inconvenience of this biosensor system is that,
here also, the polythiophene backbone redox signal is situ-
ated at a high potential, which may cause the degradation of
the immobilized bioreceptor on its surface.
So, another new approach is to functionalize the terthio-
phene by a redox group to use another redox signal rather
than the polythiophene backbone redox signal to detect the
target biomolecule. In this last approach, the terthiophene
was used only to bind the redox group on the electrode sur-
face by electropolymerization of the terthiophene moiety.
This is the case of the terthiophene functionalization by a
ferrocene (20) used to develop a cytochrome C biosensor.
Wallace et al. have covalently bonded a ferrocene ring to the
central thiophene ring in the 3′ position by a π-conjugated
linker, allowing to make a redox-active polythiophene film
from the ferrocene-functionalized terthiophene alone. In-
deed, the π-conjuguated linker allows an electronic commu-
nication between the polythiophene backbone and the
ferrocene group, so that the insulation of the polythiophene
backone is avoided. The advantage of this last approach is
the use of a redox signal from another redox group as elec-
tronic transductor (i.e., ferrocene ring) rather than from the
polythiophene moiety of the poly(terthiophene 3′-ferrocene)
film. Effectively, the ferrocene redox potentials were clearly
′′
(2,2′:5′,2 -terthiophene) (monomer 4a), the 3′-(3-chloro-4-
′′
amino-β-styryl)-(2,2′:5′,2 -terthiophene) (monomer 4b),
′′
the 3′-(3-methoxy-4-amino-β-styryl)-(2,2′:5′,2 -terthiophene)
(monomer 4c), and the 3′-(2-methoxy-5-amino-β-styryl)-
(2,2′:5′,2 -terthiophene) (monomer 4d). This new strategy to
′′
functionalize the terthiophene by an amino β-styryl group
gives a new ECP having the following properties:
(i) the ECP from amino β-styryl terthiophene monomers
can be electrodeposited on an electrode surface by
electropolymerization of the terthiophene moiety in
which the redox signal of the aminophenyl moiety is
used as an electronic transducer signal to detect and to
measure target biomolecules. Also, we have introduced
Y substitutes having different inductive, steric, and
mesomeric effects on the aminophenyl ring to study
their influences on its redox-activity to lower the
aminophenyl anodic potential as low as possible towards
0 V vs. Ag/AgCl.
(ii) the π-conjuguated linker allows an electronic communi-
cation between the aminophenyl moiety and the poly-
thiophene backbone to avoid an insulating effect on the
polythiophene backbone. Consequently, copolymeriza-
tion to obtain a redox-active polymer film is avoided.
(iii) the presence of free primary amine groups on the ECP
film surface permits further biological functionalization
of its surface by covalently bonding various biorecep-
tors. This biosensor system can be used for various tar-
gets biomolecules.
Experimental
Materials and instrumentation
All reagents were purchased from the Sigma-Aldrich
Chemical Company (Canada) and used as received: 3-methyl-
4-nitrobenzyl bromide, triphenylphosphine (PPh3), 3-chloro-
benzyl chloride, benzene, 1,8-diazabicyclo(5.4.0)undec-7-
ene (DBU), iron powder (Fe), ammonium chloride (NH4Cl),
acetic acid glacial (AcOH), sulfuric acid fuming (H2SO4),
nitric acid (HNO3), 2-methoxy-5-nitrobenzyl bromide, tin(II)
chloride-dihydrate (SnCl2·2H2O), 5-methyl-2-nitrophenol,
© 2008 NRC Canada

1012
Can. J. Chem. Vol. 86, 2008
dimethyl sulfate ((CH3)2SO4), dibenzoyl peroxide, N-
hydroxysuccinimide (NHS), N-bromosuccinimide (NBS),
magnesium sulfate (MgSO4), sodium sulfate anhydrate
(Na2SO4), ethyl-3-(3-dimethylamino-propyl) carbodiimide
(EDC), Biotin, and tetrabutylammonium perchlorate (TBAP,
electrochemical grade). Dichloromethane (DCM, HPLC
grade), avidin from egg white, and the phosphate buffer sa-
line (PBS 0.1 mol/L) of pH 7.4 at 25 °C were purchased
from Fischer Scientific (Canada). All experimental solutions
were prepared using double-distilled water obtained from a
Milli-Q water system.
1039, 959, 844, 827, 753, 721, 706 cm–1. 1H NMR (acetone-
d6, 400 MHz) δ: 8.05 (dd, 1H, H5p), 7.75 (s, 1H, H4′), 7.68–
7.63 (m, H6p, Hb, H5), 7.62–7.58 (2s, 1H, H2p), 7.53 (dd, 1H,
H ), 7.43–7.35 (m, H , H , H ), 7.25 (dd, 1H, H ), 7.15
′′
′′
5
3
4
(dd, 1H, H ), 2.6 (s,23H, –C3H ). 13C NMR (acetone-d6,
′′
100.6 MHz4) δ: 149, 143, 137, 136.8, 136.7, 135, 134.9,
133.7, 131.5, 129.5, 129.1, 128.9, 128.3, 128.1, 126.5, 126,
125.7, 125.5, 125, 123.3, 20 (–CH3). MS m/z: 410 (MH+).
3
′′
3′-(3-Chloro-4-nitro-␤-styryl)-(2,2′:5′,2 -terthiophene) (3b)
Colour: orange. Solid, yield: 20%, mp 115 °C. IR (KBr):
3100, 3063, 2960, 2854, 1588, 1515 and 1337 (NO2), 1042,
Nuclear magnetic resonance (NMR) spectra were mea-
sured at 400.13 MHz (1H) and 100.6 MHz (13C) on a Bruker
ARX 400. All NMR spectra were recorded in deutero-
acetone solutions with tetramethylsilane as reference. Sig-
nals are described in terms of chemical shifts, multiplicity,
and assignment. The following abbreviations were used: s
(singlet), d (doublet), dd (doublet of doublet), m (multiplet),
and b (broad). In the NMR spectrum interpretation, the
phenyl protons will have a letter p accompagnying the posi-
tion number to avoid the confusion with the protons of the
first thiophene ring (e.g., H5p corresponds to the proton of
the phenyl in 5 position). Mass measurements were per-
formed on a LC–MSD–TOF instrument from Agilent Tech-
nologies in positive electrospray mode. Either protonated
molecular ions (M + H)+ or sodium adducts (M + Na)+ were
used for peak assignment. The IR spectra were recorded on
a NEXUS 670 FTIR apparatus over the 600–4000 cm–1
range. The melting points were measured using a Fisher–
Johns melting point apparatus. The measuring set-up for the
all electrochemical experiments consisted of a three-
electrode system in which a glassy carbon electrode or a
platinum electrode, Ag/AgCl (saturated KCl) electrode, and
a platinum wire (0.5 mm of diameter) or platinum foil (di-
mension 1 cm × 1 cm, 0.025 mm2 of thickness) were used as
the working, the reference, and the auxiliary electrode, re-
spectively. The cyclic voltammograms (CVs) and the square-
wave voltammogram (SWVs) were recorded using a
Solartron-SI 1287 potentiostat/galvanostat and a EG & G
Princeton Applied Research 273 A one, respectively. The
measurements were carried out in a 0.1 mol/L TBAP–DCM
solution at room temperature.
1
953, 844, 831, 815, 746, 683 cm–1. H NMR (acetone-d6,
400 MHz) δ: 8.15 (d, 1H, H5p), 7.85 (d, 1H, H2p), 7.8 (dd,
1H, H6p), 7.75 (s, 1H, H4!), 7.68 (d, 1H, Hb), 7.65–7.5 (m,
2H, H , H ), 7.45–7.38 (m, 3H, H , H , H ), 7.25 (dd, 1H,
′′
′′
3
5
a
H ), 75.15 (dd, 1H, H ). MS m/z: 430 3(MH+).
′′
4
4
′′
3′-(3-Methoxy-4-nitro-␤-styryl)-(2,2′:5′,2 -terthiophene) (3c)
Colour: orange. Solid, yield: 15%, mp 148 °C. IR (KBr):
3100, 3082, 2965, 2930, 2843, 1595, 1504 and 1343 (NO2),
1
1086, 1021, 830, 704 cm–1. H NMR (acetone-d6, 400 MHz)
δ: 7.89 (d, 1H, H5p), 7.79 (d, 1H, Hb), 7.72 (s, 1H, H4′), 7.6
(d, 1H, H ), 7.55–7.5 (m, 3H, H , H , H ), 7.44 (d, 1H,
′′
H ), 7.432–p7.34 (m,2H, H , H ), a7.29 (dd, 1H, H ), 7.15
5
5
′′
6p
3
3
4
(dd, 1H, H ), 4.04 (s, 3H, OCH ). 13C NMR (acetone-d6,
′′
4
3
100.6 MHz) δ: 149, 143, 137, 136.8, 136.7, 135, 134.9,
133.7, 131.5, 129.5, 129.1, 128.9, 128.3, 128.1, 126.5, 126,
125.7, 125.5, 125, 123.3, 20 (–CH3). MS m/z: 426 (MH+).
′′
3′-(2-Methoxy-5-nitro-␤-styryl)-(2,2′:5′,2 -terthiophene) (3d)
Colour: yellow. Solid, yield: 61%, mp 115 °C. IR (KBr):
3108, 3082, 2969, 2939, 2843, 1582, 1513 and 1339 (NO2),
1
1095, 1026, 826, 743, 704 cm–1. H NMR (acetone-d6, 400
MHz) δ: 8.38(d, 1H, H6p), 8.19 (dd, 1H, H4p), 8.01 (d, 1H,
H ), 7.8 (s, 1H, H ),7.65 (m, 2H, H , H ), 7.35 (d, 1H, H ),
′′
4′
5
5
a
7.b25 (m, 3H, H , H , H ), 7.05 (m, 2H, H H ), 4.04 (s,
′′
′′
4
3H, OMe). MS3mp /z: 34263(MH+).
4,
General procedure for the synthesis of amino ␤-styryl
terthiophene monomers (4a, 4b, 4c, and 4d)
General procedure for the synthesis of nitro ␤-styryl
terthiophene (3a, 3b, 3c, and 3d)
Amino-β-styryl terthiophene compounds 4a and 4b were
obtained by the reduction of the nitro group of the corre-
sponding compounds 3a and 3b using iron powder (Fe) in
presence of NH4Cl.
A mixture of a terthiophene aldehyde (1) prepared accord-
ing to the literature method (24) (1 mmol), the triphenyl-
phosphonium bromide compounds (2a, 2c, and 2d) or
triphenylphosphonium chloride compound (2b) (1.2 mmol),
and DBU (1.2 mmol) in dichloromethane (15 ml) was heated
under reflux. After 8 h, the reaction mixture was diluted
with dichloromethane (100 ml) and washed with a 1 mol/L
solution of HCl (2 × 40 ml), 10 % sodium bicarbonate solu-
tion (40 ml), and water (60 ml). The organic layer was dried
and concentrated to give a crude organic solid. The pure
product was obtained by chromatography on a silica-gel col-
umn using a dichloromethane/hexane solution (1:1) as eluent.
To nitrophenyl compounds (1 mmol) in THF/EtOH/H2O
with the ratio of 3:3:2 were added iron powder (3 mmol) and
NH4Cl (0.6 mmol). The reaction was stirred at 85 °C for 1 h,
cooled to room temperature, and filtered through a Celite
column. The filter cake was washed with CHCl3, and the fil-
trate was concentrated to a low volume, diluted with CH3Cl,
and washed with H2O. The organic extracts were dried over
MgSO4 and filtered. After removing the solvent, the com-
pounds were separated by a silica column with EA:CH2Cl2
ratio of 2:1, then pure EA. After purification, the compounds
were obtained with good yields. In this reduction process, to
improve the solubility of the materials, the THF was used as
co-solvent
′′
3′-(3-Methyl-4-nitro-␤-styryl)-(2,2′:5′,2 -terthiophene) (3a)
Colour: orange. Solid, yield: 71%, mp 148 °C. IR (KBr):
3104, 3073, 2965, 2906, 1595, 1511 and 1333 (NO2), 1078,
© 2008 NRC Canada

Mehenni and Dao
1013
′′
sis of 2a and 2d. In this reaction, the 2b and 2b′ were given.
Because of the difficulty of the separation, the mixture was
not separated before used for the Wittig reaction. After the
Wittig reaction, the pure desired isomer 3b was isolated by
TLC plates of silica gel (20 × 20 cm, thickness of 2000 µm),
with cyclohexane/acetone (10:1) as eluent, from the reaction
mixture following removal of the major isomer (3b′) by
recrystallization from dichloromethane/ether. The ylide 2c
was synthesized according to the literature method (26) to
prepare the 3-methoxy-4-nitrobenzyl bromide and then ac-
cording the synthesis of 2a and 2d.
3′-(3-Methyl-4-amino-␤-styryl)-(2,2′:5′,2 -terthiophene) (4a)
Colour: brown. Liquid, yield: 100%. IR (NaCl): 3473 and
3373 (NH2), 3104, 3073, 2965, 2926, 2873, 1604, 1260,
1082, 1039, 960, 821, 760, 695 cm–1. H NMR (acetone-d6,
1
400 MHz) δ: 7.66 (dd, 1H, H5′′), 7.5 (dd, 1H, H5), 7.43–7.36
(m, 2H, H , H ), 7.34 (d, 1H, H ), 7.31 (d, 1H, H ), 7.25–
′′
7.18 (m, 23H, H3 , H ), 7.16 (s, 1aH, H ), 7.14 (m, 2H, H ,
b
′′
4
4′
H6p), 6.7 (d, 1H4, H2p), 3.8 (sl, 2H, –NH2), 2.8 (s, 3H, –CH53p).
MS m/z: 380 (MH+, 100%).
′′
3′-(3-Chloro-4-amino-␤-styryl)-(2,2′:5′,2 -terthiophene) (4b)
Colour: brown. Liquid, yield: 100%. IR (NaCl): 3465 and
3373 (NH2), 3104, 3069, 2960, 2921, 2852, 1613, 1265,
Electrochemistry
1095, 1043, 956, 817, 760, 700 cm–1. H NMR (acetone-d6,
1
Electrochemical polymerization of monomers 4a, 4b, 4c,
and 4d
400 MHz) δ: 7.62 (dd, 1H, H ), 7.5 (dd, 1H, H ), 7.45
′′
(d,1H, H ), 7.37 (dd, 1H, H ), 57.33 (dd, 1H, H ), 7.31 (d,
5
′′
1H, H ), b7.22 (m, 2H, H , H ), 7.19 (s, 1H, H ), 7.14 (m,
3
3
The monomers 4a, 4b, 4c, and 4d were electrodeposited
on a polished glassy carbon electrode by electropolymeri-
zation of 5 mmol/L of the monomer in a 0.1 mol/L TBAP–
DCM solution by potential cycling between 0.0 and 1.1 V
(vs. Ag/AgCl) at 10 mV s–1. After the polymerization, the
electrode surface was rinsed with DCM to remove the excess
monomer. Post-polymerization cyclic voltammograms and
square-wave voltammograms of homopolymers 4a, 4b, 4c,
and 4d were investigated in 0.1 mol/L TBAP–DCM solu-
tion.
The neutral polymers were characterized by reflectance
FTIR experiments and X-ray photoelectron spectroscopy
(XPS). FTIR spectra of polymers films on gold-coated glass
electrodes were recorded on NEXUS 670 FTIR over the
range 600–4000 cm–1. XPS examination were carried out on
the polymers films grown on ITO-coated glass electrodes us-
ing a Escalab 220i XL spectrometer.
′′
2H, Ha2p, H6p), 6.9 (d, 14H, H45p), 3.85 (sl, 2H,4–′ NH2). MS
m/z: 400 (MH+, 100%).
For the amino-β-styryl terthiophene compounds 4c and 4d,
the nitro groups of 3c and 3d compounds were reduced by
SnCl2·2H2O. To nitrophenyl compounds (1 mmol) in
EtOH/CH2Cl2 with the ratio of 5:2 were added SnCl2·2H2O
(12 mmol). The reaction was stirred at 90 °C for 17 h,
cooled to room temperature, and then 1 g sodium bicarbon-
ate was added. The reaction mixture was stirred during
30 min and then diluted with dichloromethane (100 ml) and
filtered through Celite. The filtrate was washed with 5% so-
dium bicarbonate solution (2 × 100 mL), washed with H2O
(1 × 100 mL), and then dried over anhyd. Na2SO4. After re-
moving the solvent, the product was obtained by column
chromatography (silica gel) with ethyl acetate as eluent.
′′
3′-(3-Methoxy-4-amino-␤-styryl)-(2,2′:5′,2 -terthiophene) (4c)
Surface biofunctionalization
Colour: brown. Liquid, yield: 74%. IR (NaCl): 3481 and
3373 (NH2), 3108, 3073, 2973, 2930, 2873, 1617, 1252,
Poly(4b)- and poly(4d)-coated electrodes were immersed
in a 0.1 mol/L phosphate buffer saline (PBS) solution (pH 8)
containing 0.05 mol/L EDC and 0.05 mol/L biotin for 24 h
at 40 °C. The biotin was immobilized through the formation
of covalent bond with free primary amine groups on the
polymer surface. These electrodes were then rinsed thor-
oughly with the same buffer to remove the non immobilized
biotin, and their cyclic voltammograms (CVs) and square-
wave voltammograms (SWVs) were recorded. Finally, this
poly(4c)/ and poly(4d)/biotin-modified electrode were incu-
bated in a 0.1 mol/L phosphate buffer saline (PBS) solution
of 3.10–4 mol/L avidin for 15 min at room temperature to
block active sites of avidin by a specific interaction with bio-
tin. These electrodes were removed, rinsed with the same
buffer to remove the non reacted avidin with biotin, and al-
lowed to dry. Then, their CVs and SWVs were recorded.
1126, 1056, 834, 695 cm–1. H NMR (acetone-d6, 400 MHz)
1
δ: 7.33 (s, 1H, H ), 7.6–7.55 (m, 2H, H , H ), 7.52 (d, 1H,
′′
4 ′
5
Hb), 7.53–7.2 (m, 5H), 7.2–7.1 (m, 3H, phen5yl), 3.95 (s, 3H,
OCH3), 3.6 (sl, 2H, NH2). MS m/z: 396 (MH+, 100%).
′′
3′-(2-Methoxy-5-amino-␤-styryl)-(2,2′:5′,2 -terthiophene) (4d)
Colour: brown. Liquid, yield: 54%. IR (NaCl): 3443 and
3360 (NH2), 3104, 3069, 2960, 2926, 2856, 1617, 1265,
1100, 1030, 808, 756, 700 cm–1. H NMR (acetone-d6, 400
1
MHz) δ: 7.78 (dd, 1H, H ), 7.68 (dd, 1H, H ), 7.63 (dd, 1H,
′′
5
5
H ), 7.5 (dd, 1H, H ), 7.42 (s, 1H, H ), 7.4 (d, 1H, H ),
′′
7.333 (d, 1H, H ), 7.23–7.15 (m, 2H, H4,′ H ), 6.95 (d, 1H,
3
a
′′
H6p), 6.8 (dd, 1H, H3p), 6.64 (dd, 1H, H44p), 4.3 (sl, 2H,
b
4
NH2), 3.8 (s, 3H, OMe). MS m/z: 396 (MH+, 100%).
General procedure for the synthesis of ylides
compounds 2a, 2b, 2c, and 2d
Results and discussion
A mixture of the 3-methyl-4-nitrobenzyl bromide (1
mmol) or 2-methoxy-5-nitrobenzyl bromide (1 mmol) and
PPh3 (1.2 mmol) in benzene was heated under reflux. After
3 h, the pure product 2a or 2d was recuperated as a white
solid by removal of the solvent. The ylide 2b was synthe-
sized from the commercially available 3-chlorobenzyl chlo-
ride according to the literature method (25) to prepare the 3-
chloro-4-nitrobenzyl chloride and then according the synthe-
Synthesis of amino ␤-styryl terthiophene monomers (4a,
4b, 4c, and 4d)
Scheme 1 shows the synthetic routes for amino β-styryl
terthiophene monomers (4a–4d). The terthiophene aldehyde
precursor (1) was prepared according to ref. 24. Starting
from compound 1, two steps are necessary to prepare the
monomers 4a, 4b, 4c, and 4d. The first step is a Wittig reac-
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Can. J. Chem. Vol. 86, 2008
Scheme 1. The synthesis of the monomers.
tion to make the conjugated link between the aminophenyl
moiety and the terthiophene. Compound 1 was treated with
various ylide compounds (2a–2d) in DCM in presence of
DBU to obtain the nitro β-styryl terthiophene compounds
(3a–3d). The second step is a reduction of the nitro group of
the compounds 3a, 3b, 3c, and 3d to provide the final amino
β-styryl terthiophene monomers (4a–4d). Iron powder (Fe)
was used as reducing agent for 3a and 3b and tin(II) chlo-
ride dihydrate (SnCl2·2H2O) for 3c and 3d. The ylide com-
pounds (2a–2d) were synthesized according to Scheme 2.
The ylide compounds 2a and 2b were prepared from com-
mercially available materials, the 3-methyl-4-nitrobenzyl
bromide (a) and the 2-methoxy-5-nitrobenzyl bromide (d),
which were treated by PPh3 in benzene. The ylide 2b was
prepared according to ref. 27 to synthesize the 3-chloro-4-
nitrobenzyl chloride, which was treated by PPh3 in benzene.
The ylide 2c was synthesized according to ref. 28 to prepare
the 3-methoxy-4-nitrobenzyl bromide (c), which was treated
by PPh3 in benzene.
cyclic voltammograms show the expected increase in current
with increasing number of potential cycles, indicating the
growth of electronic conducting polymers (ECP). The elec-
trochemical polymerization of monomers 4a, 4b, 4c, and 4d
results in the formation of a black uniform polymer film on
the electrode surface.
Electrochemical characterization of poly(amino ␤-styryl
terthiophene) films
Figure 2 shows cyclic voltammograms (CVs) of poly(4a),
poly(4b), poly(4c), and poly(4d) films at scan rate 5 mV s–1
by potential cyclic between 0 and 1.0 V (vs. Ag/AgCl). The
scanning from 0 to 1.0 V shows clearly two anodic peaks for
poly(4c) and poly(4d) films, which we named a and c, and
which we attributed to the oxidation of the aminophenyl
moiety and to the polaronic oxidation of the polythiophene
backbone, respectively. Indeed, it is reported that the
aminophenyl moiety can be oxidized to give a radical cation
(27). The oxidation removes an electron from the free elec-
trons doublet of the nitrogen in its neutral state (NH2), to
give a radical cation nitrogen (NH2C+). We were able to at-
tribute the origin of the peaks a and c compared with the cy-
clic voltammogram of a polyterthiophene film prepared in
the same conditions, and also by using literature results for
polyterthiophene for which it is reported (28) that the
polaronic oxidation peak is situated towards 1.05 V vs. SCE
Synthesis of poly(amino ␤-styryl terthiophene)
(poly(4a), poly(4b), poly(4c), and poly(4d))
Monomers 4a, 4b, 4c, and 4d were electrodeposited on
glassy carbon electrodes by cyclic voltammetry between 0
and 1.1 V (vs. Ag/AgCl). Figure 1 shows the CVs for the
formation of poly(4d) film on a glassy carbon electrode. The
© 2008 NRC Canada

Mehenni and Dao
1015
Scheme 2. Synthesis of the ylide compounds.
Fig. 1. Growth of poly(4d) by repetitive-scan cyclic voltammetry
on a 7.068 mm2 glassy carbon electrode between 0.0 and 1.1 V,
auxiliary electrode: Pt foil, reference electrode: Ag/AgCl, scan
rate: 10 mV s–1m, 5 mmol/L monomer 4d, 0.1 mol/L TBAP–DCM.
Fig. 2. Cyclic voltammogram of poly(4a), poly(4b), poly(4c),
and poly(4d) films between 0.0 and 1.0 V, scan rate: 5 mV s–1 on
a 7.068 mm2 glassy carbon working electrode, auxiliary electrode:
Pt wire, reference electrode: Ag/AgCl, 0.1 mol/L TBAP–DCM.
(corresponding to 1.09 V vs. Ag/AgCl). For poly(4a) and
poly(4b) films, we can only see the peak a and an oxidation
slope attributed to the polythiophene backbone oxidation.
When the scanning direction is inverted from 1.0 to 0.0 V
vs. Ag/AgCl, we see two cathodic peaks, d and b, for
poly(4c) and poly(4d) films, which we attributed to the re-
duction of the polythiophene backbone and to the amino-
phenyl moiety reduction, respectively. The top of the peak b
will be more clearly visible by SWV for the poly(4b) film.
For the poly(4a) film, the last peaks are not distinctly visible
because they are overlapping. So, the introduction of Y sub-
stitutes in the aminophenyl ring has for consequence to
make the aminophenyl redox couple (a/b) visible and differ-
ent from the polythiophene backbone redox couple (c/d). It
lowers its anodic potential, in the first place situated at 0.98
V vs. Ag/AgCl (corresponding to the aminophenyl anodic
potential (29)) towards a lower potential, especially when
Y = OCH3, which is a strong electronic donor and for which
the aminophenyl anodic potential is slightly below 0.5 V vs.
Ag/AgCl.
The square-wave voltammetry allows to eliminate the
current overload (capacitive current Ic) visible in cyclic
voltammetry (30–32). In other words, we can subtract Ic
from the total current measured. Consequently, we used it to
see clearly the redox couple (a/b) attributed to the
aminophenyl moiety of poly(4a), poly(4b), poly(4c), and
poly(4d) films. The square-wave voltammograms of poly-
mer films are reported in Fig. 3. During the oxidation scan-
ning from 0.0 to 1.0 V vs. Ag/AgCl, we can observe two
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1016
Can. J. Chem. Vol. 86, 2008
Fig. 3. Square-wave voltammograms of poly(4a), poly(4b),
poly(4c), and poly(4d) films between 0.0 and 1.0 V on a 7.068 mm2
glassy carbon working electrode, auxiliary electrode: Pt foil, ref-
erence electrode: Ag/AgCl, 0.1 mol/L TBAP–DCM.
Fig. 4. (A) XPS spectrum of the poly(4d) film and (B) FTIR
spectrum of the poly(4b) film, grown on ITO-coated glass and
gold-coated glass, respectively, at room temperature.
oxidation peaks, a and c, for the poly(4d) film. The anodic
peak a situated towards 0.5 V vs. Ag/AgCl corresponds to
the oxidation of the aminophenyl moiety, and the anodic
peak c situated towards 0.9 V vs. Ag/AgCl corresponds to
the polaronic oxidation of the polythiophene backbone. But
for poly(4a), poly(4b), and poly(4c) films, we observe only
the anodic peak a, situated towards 0.6 V vs. Ag/AgCl for
poly(4a) and poly(4b), and towards 0.5 V vs. Ag/AgCl for
poly(4c). In higher potentials, we observe an oxidation slope
attributed to the oxidation of the polythiophene backbone.
During the reduction scanning from 1.0 to 0.0 V vs.
Ag/AgCl, we can observe two reduction peaks, d and b, for
the poly(4d) film, corresponding to the reduction of the
polythiophene backbone and to the aminophenyl moiety re-
duction, respectively. But for the poly(4a), poly(4b) and
poly(4c), we observe a reduction slope corresponding to the
reduction of the polythiophene backbone and the cathodic
peak b.
Biotin-modified electrodes
We have begun to investigate the possibility of using the
poly(4b) and poly(4d) films in biosensor devices in which
the redox signal of the aminophenyl moiety is used as an
electronic transducer signal to detect a target biomolecule.
We chose to begin with biotin–avidin interaction. Avidin is a
highly stable glycoprotein and is able to be specifically
bound by biotin to form an exceedingly strong complex (37).
The steps used to link the biotin to the poly(amino β-styryl
terthiophene)-coated electrode surface are summarized in
Fig. 5. This procedure has the advantage of using simple
conditions and of forming a covalent bond. The cyclic and
the square-wave voltammograms of the poly(4b)- and
poly(4d)-coated working electrode, before and after immobi-
lization of biotin on their surfaces, are reported in Fig. 6a
and Fig. 7a, respectively. We can observe for both polymer
films that the biotin immobilization causes a shift towards a
higher potential of the anodic peak initially attributed to the
oxidation of the aminophenyl moiety. After biotin immobili-
zation, the anodic peak corresponds to the oxidation of the
amidophenyl resulting on the polymer film surface. Indeed,
it is reported (29) that the amidophenyl anodic peak (Aceto-
amidophenyl EpA = + 1.24 V vs. Ag/AgCl) is higher than
that of the aminophenyl (EpA = + 0.98 V vs. Ag/AgCl).
The cyclic and the square-wave voltammograms of the
poly(4b)/ and poly(4d)/biotin-modified-coated working elec-
trode, before and after exposure to a solution of 3.10–4 mol/L
avidin in PBS 0.1 mol/L for 15 min are shown in Fig. 6b and
Fig. 7b, respectively. We can observe in both cases, i.e., by
CV or SWV, that the exposure of the poly(4b)/ or
poly(4d)/biotin-modified-coated working electrode has a
drastic effect upon its electrochemical response causing a
significant diminution of the oxidation current measured on
the anodic peak. This unusual voltammetry response after
avidin exposure is explained by the presence of avidin cov-
ering the polymer film surface and thus causing the reduc-
Spectroscopic FTIR and XPS characterization
The studies of the poly(4a), poly(4b), poly(4c), and
poly(4d) films by FTIR and XPS techniques, clearly show
that there are free primary amine groups on the polymer film
surface after electrochemical polymerization. This point
proves that the amino β-styryl moiety is not destroyed during
electropolymerization, and it does not bind anything by the
amine groups. As an example, the XPS spectrum of the
poly(4d) film and the FTIR spectrum of the poly(4b) film
are shown in Fig. 4. FTIR spectrum of poly(4b) film grown
on gold-coated glass shows two strong sharp peaks at 3373
and 3213 cm–1 (amine I band, N–H stretching vibration
(33)) corresponding to the primary amine groups on the
aminophenyl moiety, and a C–H stretching vibration of the
aromatic ring (34) is also observed at 3065 cm–1.
XPS spectrum shows the deconvoluted N1s spectrum of
the poly(4d) film surface grown on ITO-coated glass elec-
trode. The two features at 402.5 and 399.6 eV must be as-
+
cribed to a cationic nitrogen species (NH3 and NH2C+) and
an amine I species (NH2), respectively (35, 36).
© 2008 NRC Canada

Mehenni and Dao
1017
Fig. 5. Schematic representation of the immobilization of recep-
tive biomolecule (biotin) via the activation of carboxylic func-
tional groups (actived by EDC) of the biotin.
Fig. 7. (a) Square-wave voltammograms of poly(4d)-coated
glassy carbon working electrode before and after immobilization
of biotin. (b) Square-wave voltammograms of poly(4d)/biotin-
modified-coated glassy carbon working electrode before and after
exposure in a solution of avidin 3.10–4 mol/L in PBS 0.1 mol/L
during 15 min, auxiliary electrode: Pt foil, reference electrode:
Ag/AgCl, 0.1 mol/L TBAP–CH2Cl2.
Fig. 6. (a) Cyclic voltammograms of poly(4b)-coated platinum
working electrode before and after immobilization of biotin.
(b) Cyclic voltammograms of poly(4b)/biotin-modified-coated
platinum working electrode before and after exposure in a solu-
tion of avidin 3.10–4 mol/L in PBS 0.1 mol/L during 15 min,
auxiliary electrode: Pt foil, reference electrode: Ag/AgCl, scan
rate: 25 mV s–1, 0.1 mol/L TBAP–CH2Cl2.
tion in the number of amidophenyl moieties able to be
oxidized at the polymer-film surface.
Conclusions
electrode, after exposure to an avidin solution, suggest that
these polymer films have potential application as biosensors.
We project to develop an avidin biosensor by using this
last system to measure an unknown avidin concentration.
Some of our results will be submitted soon. To investigate
the possibility of using the poly(amino β-styryl terthiophene)
films for various target biomolecules, we project also to
make futher functionalization on the last ECPs surfaces by
other bioreceptors.
The terthiophene was successfully functionalized by an
amino β-styryl moiety. We synthesized four new monomers
according to the Y substituent, the 3′-(3-methyl-4-amino-β-
′′
styryl)-(2,2′:5′,2 -terthiophene) (monomer 4a), the 3′-(3-
′′
chloro-4-amino-β-styryl)-(2,2′:5′,2 -terthiophene) (mono-
′′
mer 4b), the 3′-(3-methoxy-4-amino-β-styryl)-(2,2′:5′,2 -
terthiophene) (monomer 4c), and the 3′-(2-methoxy-5-
′′
amino-β-styryl)-(2,2′:5′,2 -terthiophene) (monomer 4d).
The homopolymers from monomers 4a, 4b, 4c, and 4d have
been successfully electrosynthesized. The electrochemical
studies of these polymer films display both the aminophenyl
moiety redox couple as well as the polythiophene backbone.
The cyclic and square-wave voltammogram responses of the
poly(4b)/ and poly(4d)/biotin-modified film on working
Acknowledgements
This work was supported by the National Institute of the
Scientific Research (INRS) and the Natural Sciences and
Engineering Research Council of Canada (NSERC).
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Can. J. Chem. Vol. 86, 2008
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© 2008 NRC Canada