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C. Gondran et al. / Electrochemistry Communications 12 (2010) 311–314
1H NMR (250 MHz, CDCl3): d ppm = 6.73 (2H, s); 6.21 (2H, s);
from 50 to 0.1 Hz at ꢀ0.45 V vs. SCE with an alternating voltage
of 5 mV rms.
4.12 (2H, t, J = 6.3 Hz); 3.35 (2H, t, J = 6.3 Hz); 2.30 (2H, quint,
J = 6.3 Hz).
The permeability of the poly 1 was determined by using rotat-
ing-disc electrode (RDE) experiments carried out at different rota-
tion rates in 0.1 mol Lꢀ1 phosphate buffer (pH 7) containing
hydroquinone (2 ꢂ 10ꢀ3 mol Lꢀ1). Stationary current was recorded
between 0 and 0.5 V vs. SCE.
13C NMR (62.5 MHz, CDCl3): d ppm = 120.69 (CH); 108.49 (CH);
47.10 (CH2); 34.24 (CH2); 30.41 (CH2).
N-(3-pyrrol-1-ylpropyl)-4-(40-pyridyl) pyridinium was synthe-
sized as follows: a mixture of bromo-pyrrole (0.4715 g, 2.5 mmol)
and 2 equivalents of 4,40-bipyridine (0.7819 g, 5.0 mmol) was stir-
red at 85 °C for 20 h in DMF (5 mL). After solvent evaporation, H20
was added and the aqueous layer was extracted three times with
CH2Cl2. After evaporation of water, the mono-alkylated product
was dried under vacuum and thus obtained in 87% yield
(0.7536 g, 2.19 mmol).
2.3. Avidin-alkaline phosphatase immobilization and characterization
UV–vis measurements were obtained using a Cary 1 absorption
spectrophotometer. The poly1-Av-AP electrodes were prepared by
incubation of an electrode modified by a film of poly (N-(3-pyrrol-
1-ylpropyl)-N0-(3-propyl biotin)-4, 40-bipyridinium) (poly1,
1H NMR(250 MHz, DMSO-d6): d ppm = 9.15 (2H, d, J = 6.8 Hz);
8.87 (2H, d, J = 4.3 Hz); 8.60 (2H, d, J = 6.8 Hz); 8.03 (2H, d,
J = 4.3 Hz); 6.75 (2H, s); 5.96 (2H, s); 4.65 (2H, t, J = 6.8 Hz); 4.03
(2H, t, J = 6.8 Hz); ꢁ2.50 (2H).
C
= 1.58 ꢂ 10ꢀ9 mol cmꢀ2) with Av-AP (20
l
L, 0.5 mg mLꢀ1) for
30 min. The resulting enzyme electrodes were thoroughly washed
in stirred Tris–HCl buffer for 30 min and soaked into 3 mL of Tris–
HCl buffer (0.05 mol Lꢀ1, pH 9.4) containing p-nitrophenylphos-
phate (10ꢀ2 mol Lꢀ1) and MgCl2 (2 ꢂ 10ꢀ3 mol Lꢀ1). The Av-AP con-
centration on surface was measured by following at 410 nm the
absorbance of p-nitrophenol produced from p-nitrophenyl phos-
phate as a function of time.
13C NMR (62.5 MHz, CD3OD): d ppm = 154.41; 151.70; 146.37;
143.32; 126.91; 123.42; 121.59; 109.50; 60.53; 47.28; 33.25.
3-Bromopropyl biotin was prepared as follows: a mixture of
biotin (0.4877 g, 2.0 mmol), 3-bromopropan-1-ol (1.3880 g,
10.0 mmol) and p-toluene sulfonic acid (0.0380 g, 0.2 mmol) was
stirred and refluxed at 120 °C in toluene (12 mL) for 72 h under
nitrogen atmosphere. After toluene evaporation, the residue was
dissolved in CH2Cl2, precipitated by adding Et2O, filtered off and
purified by silica gel column chromatography (CH2Cl2/MeOH, 9/
1) providing a white solid (81% yield).
2.4. Immunoassay design
A drop (20 l
L) of avidin (0.5 mg mLꢀ1 in phosphate buffer) was
incubated for 30 min onto the poly1. After rinsing with phosphate
buffer, the resulting modified electrodes were incubated for 20 min
1H NMR(250 MHz, CDCl3): d ppm = 5.27 (1H, s); 4.92 (1H, s);
4.52 (1H, m); 4.33 (1H, m); 4.21 (2H, t, J = 6.3 Hz); 3.47 (2H, t,
J = 6.7 Hz); 3.17 (1H, m); 2.93 (1H, dd); 2.74 (1H, d, J = 12.7 Hz);
2.34 (2H, t, J = 7.1 Hz); 2.18 (2H, quint, J = 6.3 Hz); 1.71–1.44 (6H,
m).
with 20 lL of biotinylated Cholera Toxin B Subunit (CTB)
(0.5 mg mLꢀ1 in phosphate buffer). The resulting electrodes were
rinsed and washed once with phosphate buffer for 10 min. The
analyte, anti-Cholera Toxin
B Subunit (anti-CTB) antibody
(0.5 mg mLꢀ1), was then spread on the resulting electrode surface
for 20 min.
The biotinylated monomer (1) was synthesized as follows: a
stoichiometric amount of 3-bromopropyl biotin (0.1434 g,
0.39 mmol) and N-(3-pyrrol-1-ylpropyl)-4-(40-pyridyl) pyridinium
(0.1248 g, 0.39 mmol) was stirred at 60 °C in EtOH (5 mL) for
12 days. The reaction mixture was cooled at room temperature
and the ethanol was evaporated under vacuum. The residue was
dissolved in MeOH (1 mL), precipitated by adding CH2Cl2 and fil-
tered off. After anionic exchange, 1 with BFꢀ4 counter anions was
thus obtained in 45% yield.
3. Results and discussion
In this study a novel electropolymerizable biotin-labeled violo-
gen functionalized by a pyrrole group (Fig. 1), was prepared by
reaction of 3-bromoethyl biotin with N-(3-pyrrol-1-ylpropyl)-4-
(40-pyridyl) pyridinium and characterized by 1H NMR and mass
spectrometry. The 3-bromoethyl biotin was prepared by esterifica-
tion of 3-bromopropanol and biotin. The electrochemical behav-
iour of biotinylated pyrrole–viologen 1 (2 mM) was investigated
in CH3CN + 0.1 mol Lꢀ1 nBu4NClO4 (Fig. 2A). Upon reductive scan-
ning, the monomer exhibits two successive reversible peak sys-
1H NMR(250 MHz, CD3OD): 9.34 (2H, d); 9.11 (2H, d); 8.70 (2H,
d); 8.59 (2H, d); 6.69 (2H, s); 6.00 (2H, s); 4.76 (2H, t); 4.49 (1H, m);
4.32–4.16 (4H, m); 3.20 (1H, m); 2.92 (1H, m); 2.73–2.62 (4H, m);
2.49 (2H, t); 2.29 (2H, t); 1.64–1.29 (6H, m); 1.18 (2H, t). MS (ESI):
m/z = 628.1 ([M2++Brꢀ]+) and 274.8 (M2+).
tems at ꢀ0.70 V
(D
Ep = 0.06 V) and ꢀ1.12 V
(DEp = 0.06 V)
corresponding to the successive one electron reduction of the viol-
ogen group (V2+/Vꢃ+ and Vꢃ+/Vꢃꢃ) [9,10]. Upon oxidative scanning,
the cyclic voltammogram displays an irreversible peak at 1.0 V
reflecting the oxidation of the pyrrole group. This potential value
is similar to those previously reported for N-alkylpyrroles [11].
Electropolymerization of 1 was performed by controlled poten-
tial electrolysis (0.1 mC) at 0.85 V. Upon transfer into
CH3CN + 0.1 M nBu4NClO4 solution free of monomer, the cyclic vol-
2.2. Electrochemical instrumentation and procedures
Electrochemical investigations were performed under an argon
atmosphere in a conventional three-electrode cell with a potentio-
stat EG&G Princeton Applied Research 273 A in conjunction with a
Kipp and Zonen BD90 XY/t recorder. A 10ꢀ2 mol Lꢀ1 Ag+/Ag in
CH3CN electrode and a saturated calomel electrode (SCE) were
used as reference electrode in CH3CN and aqueous media, respec-
tively. The working electrodes were platinum or glassy carbon
disks (diameters 5 and 3 mm) polished with 2
(MECAPREX Press PM).
lm diamond paste
O
Electrochemical Impedance Spectroscopy (EIS) was carried out
with an Autolab potentiostat 100 (Eco Chemie, Utrecht, The Neth-
erlands) using FRA software. A ZView software (Scribner Associates
Inc.) was used to simulate the data thus obtained using an appro-
priate equivalent electrical circuit. All experimental impedance
spectra were recorded by immersing the modified electrode into
phosphate buffer (0.1 mol Lꢀ1, pH 7). The frequency sweep was
HN
NH
N
N
N
O
S
O
BF4
BF4
Fig. 1. Structure of biotinylated pyrrole–viologen: 1.