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Immobilization of drugs and biomolecules on in situ copolymerized active ester polypyrrole

coatings for biomedical applications

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2006 Biomed. Mater. 1 235
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INSTITUTE OF PHYSICS PUBLISHING

BIOMEDICAL MATERIALS

Biomed. Mater. 1 (2006) 235241

doi:10.1088/1748-6041/1/4/009

Immobilization of drugs and biomolecules

on in situ copolymerized active ester
polypyrrole coatings for biomedical
applications
Wahid Khan, Tesfa Marew and Neeraj Kumar
Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER),
Sector 67, S A S Nagar-160062, India
E-mail: neeraj@niper.ac.in

Received 3 September 2006

Accepted for publication 17 November 2006
Published 4 December 2006
Online at stacks.iop.org/BMM/1/235
Abstract
Among the plethora of polymers being exploited and employed currently for biomedical
applications, polypyrrole as a conducting polymer holds a key position since it offers several
advantages including good specific conductivity, chemical stability, polymerizability and
compatibility with mammalian cells; however, it also suffers from a few limitations that
restrict it from being an obvious winner as a coating material. In order to overcome these
limitations, pyrrole derivatives have been tried as potential alternatives. These synthesized
derivatives facilitate the immobilization/coupling of biomolecules and drugs on to the surface
so as to improve the biocompatibility and performance of implantable medical devices. In this
work, N-succinimidyl ester pyrrole (PyNSE) has been synthesized and characterized. A
synthesized monomer was copolymerized with pyrrole (Py) in different ratios to obtain smooth
and adherent copolymer coatings on the metal surface. Results suggest that among the
different coatings obtained, pure active ester functionalized polypyrrole (PPyNSE) coating is
smoother and more adherent than its different copolymers with pyrrole. The activity of the
coating was tested by attaching BSA and a model drug (p-nitroaniline) on the surface which
indicated that the concentration of these molecules on the surface can be varied by varying the
concentration of monomers, i.e. Py and PyNSE, during electropolymerization. These active
coatings may serve as a potential platform for attaching drugs and biomolecules for various
biomedical applications.
(Some figures in this article are in colour only in the electronic version)

1. Introduction
The application of polymeric materials is expanding at a rapid
pace in diverse medical fields such as tissue engineering,
implantable medical devices, artificial organs, bone repair
and drug delivery [1]. With the recent proven success
of drug-eluting stents in interventional cardiology, local
drug delivery/device combination therapy is an upcoming
new frontier in medicine with vast social and economic
potential [2]. Owing to certain well-recognized issues and
1748-6041/06/040235+07$30.00

2006 IOP Publishing Ltd

challenges with implantable medical devices, there have been

considerable efforts to develop local drug delivery/implant
device combination therapies. Surface modified medical
devices provide additional advantages such as improved
biocompatibility, infection control, low-friction profiles
and/or controlled release of bioactive compounds. Based on
the above stated facts, their applications may be categorized
into three areas: (1) improving the therapeutic function of a
medical device with drug delivery coating, (2) enhancing the
biocompatibility of a medical device along with local drug

Printed in the UK

235

W Khan et al

delivery and (3) treating localized diseases with drug delivery

directly from an implant [3].
Among the various polymers used, conducting polymers
[4] such as polypyrrole (PPy) have gained tremendous
significance due to their ability to form thin, integral and
uniform films on surfaces which find diverse applications.
Two main areas for the use of PPy are (1) as a coating
material for metals [5], particularly stainless steel that has
been extensively used for manufacturing orthopedic and other
implantable medical devices owing to its superior mechanical
properties but still needs protection of its surface by a
strongly adhered inert coating which can enhance the implant
biocompatibility; and (2) PPy being a biocompatible [6] and
electroactive polymer, it can be used as a suitable substrate
for the manipulation of cell growth and function of numerous
tissues including bone, cartilage, skin, peripheral and spinal
nerves that respond favorably to electric fields.
In spite of this, PPy is also associated with some
limitations such as its roughness, rigidity, lack of
processablity and absence of any functional group for surface
immobilization of bioactive molecules. As an alternative,
chemical modification of PPy to active ester functionalized
polypyrrole (PPyNSE) demonstrates promising potential in
both aspects; firstly, it can work as a bioactive platform for
the immobilization of biomolecules, e.g., as a scaffold in
tissue engineering applications to improve cellular responses
resulting in cell adhesion and proliferation [6], and secondly,
electrocoating of monomers conjugated with active groups
followed by covalent attachment of drugs and biomolecules
can be made feasible. This strategy has great potential to
improve biocompatibility and for localized drug delivery from
implantable devices and is yet to be exploited.
N-substituted pyrrole is a symmetrical molecule that
forms a polymer with a regular structure. Substitution at
the N-position is versatile and straightforward and hence has
been selected to prepare the functionalized pyrrole derivative.
This paper reports on the preparation and characterization of
PPy-PPyNSE coated steel surfaces bearing surface N-ethyl
succinimidyl ester groups. Further, this active coating has been
used to show its reactivity towards the amine-group-containing
drugs and biomolecules. In addition to this, controlled
surface proportion of different coating monomers using in
situ copolymerization makes our scheme suitable for various
biomedical applications. Considering the novelty of the work,
to our knowledge the present form of the proposed strategies
has never been explored before for the immobilization of
drugs and biomolecules on the metal surface by means of
electropolymerization of active ester, though Schmidt et al
[6] have recently reported electropolymerization of carboxylic
acid monomer and its further utilization as a bioactive platform
for cell adhesion.

2. Materials
Stainless steel plates (316L SS) of medical grade were used.
Pyrrole (Py), 1-(2-cyanoethyl)pyrrole, tetrabutylammonium
tetrafluoroborate and N-hydroxy succinimide (NHS) were
purchased from Aldrich, 1-ethyl-3-(3-dimethylaminopropyl)
236

[i] KOH, H2O

[ii] NHS, EDC

CN
1

2
[iii] Copolymerization
Steel surface

N
H

R-NH2
N

N
H

HN

[iv]

O
O

N
H

4
3

Scheme 1. Reaction for preparation of protein and drug derivatized

polypyrrole.

carbodiimide hydrochloride (EDC) from Fluka, albumin

(BSA, Bovine Fraction V) and p-nitroaniline (PNA) from
s.d. fine-chem Ltd and HIMEDIA, respectively. Ultrapure
water was obtained using a SG water purification system,
Germany. Other chemicals and reagents were purchased from
local Indian sources.

3. Methods
3.1. Synthesis of the monomer
An active monomer, i.e. N-succinimidyl ester pyrrole
(PyNSE), was synthesized in a two-step process (scheme
1). In the first step (i), 1-(2-carboxyethyl)pyrrole was
synthesized according to a previously reported procedure with
slight modification [5, 7, 8]. Briefly, 1-(2-cyanoethyl)pyrrole
(1) was hydrolyzed to 1-(2-carboxyethyl)pyrrole by the
addition of 5 g (3.6 mM) of 1-(2-cyanoethyl)pyrrole to 30 ml of
6 M KOH solution. This mixture was refluxed under an inert
nitrogen atmosphere overnight until no more NH3 (g) was
evolved. The resulting product was acidified to pH 4 using
8 M HCl at room temperature and extracted five times with
ether. The organic extract was then dried over magnesium
sulphate and evaporated to dryness. The off-white crude
product was recrystallized using boiling n-heptane to get white
needle shaped crystals, which were dried under a vacuum and
characterized by FTIR and 1H NMR.
Further, in the second step (ii) synthesized 1-(2carboxyethyl)pyrrole was activated to N-succinimidyl ester
pyrrole (2). For this purpose, 0.863 g (7.5 mM) of NHS
and 1.917 g (10 mM) of EDC were dissolved in 50 ml
of double distilled water followed by the addition of 0.7 g
(5 mM) of 1-(2-carboxyethyl)pyrrole and the reaction was

Immobilization of drugs and biomolecules on in situ copolymerized active ester polypyrrole coatings for biomedical applications

carried out at ambient temperature (24 C) for 12 h [9, 10].

The white precipitate formed was collected by filtration,
washed with distilled water and dried under a vacuum. The
product obtained was characterized by FTIR, MS and 1H NMR
studies.
FTIR spectra of samples were obtained using a PerkinElmer (USA) spectrophotometer. Solid samples were directly
pressed into KBr pellets and the spectra were taken in the
transmittance range 4000400 cm1. Typically 10 scans
per spectrum were recorded at 4 cm1 resolution. For
1
H NMR analysis, samples were prepared in dutereated
chloroform (CDCl3) and analyzed using Bruker (Avane DPX
300). Tetramethylsilane, contained in CDCl3, served as shift
reference. The spectra were obtained in the chemical shift ()
value of 013 ppm. Mass spectroscopy (MS) was carried out
by a mass spectroscope (LCQ model, Finnigan MAT, UK) with
Xcalibar software using APCI (atmospheric pressure chemical
ionization) as the ionization mode.
3.2. Polymer formation via in situ copolymerization
The polypyrrole coating (3) bearing reactive N-ester
succinimidyl functional group (PPyNSE) was prepared by
in situ copolymerization (iii) of Py and the PyNSE. Polymer
formation by electropolymerization was performed in a onecompartment cell [11] using an AUTOLAB PGSTAT12
potentiostat/galvanostat. Platinum wire was used as the
counter electrode and a saturated calomel electrode (SCE)
as the reference electrode. The working electrode was 0.2 mm
thick low carbon 316L SS plates which underwent
pretreatment processes prior to electrocoating. The coated
surface area of the electrode was approximately 2 cm2.
Electrocoating was carried out using 0.04 M monomer (Py +
PyNSE) and 0.1 M tetrabutylammonium tetrafluoroborate as
electrolyte by cyclic voltammetry in MeCN using a potential
range from 0.5 to 1.2 V [5, 1214]. The scan rate was
kept at 20 mV s1 and 810 scans were performed to form a
copolymer coating on a steel surface. After each experiment,
the coated steel plates were thoroughly rinsed with MeCN
followed by methanol and dried to constant weight at 65 C
under a vacuum. The initial comonomer composition was
0:100, 25:75, 50:50, 75:25 and 100:0 for PyNSE and Py,
respectively. These coatings are abbreviated as PPyNSEx
where x stands for the initial molar fraction of PPyNSE
(x = 0, 25, 50, 75 or 100). By varying these ratios, the
effect on the surface morphology and chemical composition
of the coating was investigated by FTIR and SEM. FTIR of the
coated samples were taken by scratching the coating
and pelleting after mixing with KBr. Scanning electron
micrographs were obtained with a Leo Electron Microscopy
LTD, Cambridge, UK.

coatings was tested and compared for their adherence to the

steel surface. In this test, a coated surface was first glued with
epoxy adhesive and attached to another uncoated stainless steel
surface, which were then glued together, pressed and dried by
applying force [17]. These dried plates were clamped on a
pull-stub assembly mounted on the texture analyzer and were
then pulled apart in the opposite direction and the tensile lap
shear strength (shear force required to remove the coating) was
calculated using the formula
Tensile lap shear strength =

Newton breaking force

.
Surface area of bond surface

3.4. Immobilization of biomolecules (BSA)

Selected batches of coated plates were further incubated with
biomolecules (BSA) in order to study specific interfacial
interaction of biomolecules with the coated surface [16]. The
chemical reaction leading to protein immobilization on the
coated plate is shown in scheme 1 (iv). For this purpose, the
active coated plates PPyNSEx (x = 25, 50, 75 or 100) were first
dipped in 10 mM PBS at pH 7.4 for 2 h to allow ion exchange.
Thereafter, these were incubated in 2 ml BSA solution
(1 mg ml1) in PBS for 20 h at room temperature [10, 15,
16, 18]. The plates were then washed with 0.02% Tween 20 in
PBS and twice with PBS followed by one wash with ultrapure
water to remove the physisorbed protein. The coated BSA
attached plates (4) were finally dried under a vacuum. In order
to estimate the amount of BSA immobilized on the surface,
the difference between the initial protein solution and that
obtained after dipping the plates was determined by measuring
the fluorescence intensity of the respective solutions. The
fluorescence intensity was measured by using a Perkin-Elmer
LS50B luminescence spectrometer with the excitation and the
emission wavelength at 280 nm and 354 nm, respectively
[19].
3.5. Reactivity of the active coating towards drugs
The PPyNSE100 active coating was studied in terms of its
reactivity at different drug concentrations [15]. The reaction
of coated PPyNSE100 with PNA (a model molecule bearing
the amino group) was conducted in buffered media. For this
purpose, PPyNSE coated plates were dipped in PNA solutions
of different concentrations (0.05, 0.1, 0.2, 0.5 mg ml1) in PBS
at room temperature for 20 h; then the plates were rinsed with
PBS to remove any trace of free unsubstituted drug. Drug
attachment was calculated by measuring the absorbance of
initial and final solution by a UV-Vis spectrophotometer at
381 nm. The dried coating was then characterized by FTIR.

4. Results and discussion

3.3. Pull-off adhesion test
It is important for any medical device that prepared coatings
must be adherent to the surface. The adhesion strength of
different copolymer coatings was determined by a special labmade adhesion test device fitted in a TA-XT2i texture analyzer
[15, 16]. The adhesion strength of different copolymer

4.1. Synthesis of the monomer (PyNSE)

Initially, 1-(2-carboxyethyl)pyrrole was obtained and
characterized.
FTIR (KBr) spectra showed a peak at
1713.4 cm1 corresponding to carbonyl group of acid and
absence of a peak near 2240 cm1 corresponding to the
237

W Khan et al

Figure 1. FTIR spectra of the PPyNSE-PPy coated surfaces: (A) PPyNSE100, (B) PPyNSE75 and (C) PPyNSE50. The FTIR spectra clearly
show that peak intensity corresponding to the succinimidyl ester group and pyrrolidinone group of succinimide decreased with an increase
in pyrrole composition.

nitrile group present in the starting material confirming

the complete conversion of 1-(2-cyanoethyl)pyrrole to 1-(2carboxyethyl)pyrrole. 1H NMR (CDCl3, ppm) spectra showed
the peaks at 2.82 (t, 2H, CH2COO); 4.19 (t, 2H, CH2CH2);
6.14 (d, 2H, CH pyrrole); 6.66 (dd, 2H, CH pyrrole) that
complied with the true structure of 1-(2-carboxyethyl)pyrrole.
Further, coupling reaction between 1-(2-carboxyethyl)pyrrole
and NHS resulted in N-succinimidyl ester pyrrole as a white
crystalline solid with a final yield of 81%. The FTIR spectra
showed peaks at 1738.9 corresponding to the succinimidyl
ester group and at 1778.8 and 1811.9 cm1 for pyrrolidinone
group of succinimide, and peaks at 1207 and 1068 cm1
for N-O and C-O stretching, respectively. Mass spectra
of the compound showed a base peak at 236.9 Da which
corresponds to the M+1 peak of N-succinimidyl ester pyrrole.
1
H NMR (CDCl3, ppm): 2.84 (s, 4H, CH2CON); 3.07 (t, 2H,
CH2COO); 4.30 (t, 2H, CH2N); 6.16 (d, 2H, CH pyrrole); 6.69
(dd, 2H, CH pyrrole) also confirmed the formation of product
(2). NSE derivatization of 1-(2-carboxyethyl)pyrrole was
selected due to its well-known reactivity with amines under
mild conditions which opens the way for covalent attachment
of amine containing compounds on the coated surfaces.

to Py repeating units was also observed. The FTIR spectra

of different copolymer compositions (figure 1) clearly showed
as PyNSE proportion decreased intensity of peaks at 1738.9,
1778.8 and 1811.9 cm1 for both succinimidyl ester and
pyrrolidinone groups were reduced accordingly. This clearly
indicates that the proportion of copolymer composition in the
coating varies with initial feed ratio of the monomers.
It has been found that tissue response to any implantable
medical device is influenced by the surface topography
(roughness, texture and porosity) of that medical device;
therefore, surface smoothness is the most desired feature of
any implantable medical device which dictates the tolerability
of the material in a tissue environment. In agreement with this,
the surface morphology of the coated material was evaluated
by SEM (figure 2). Results clearly indicate that the coating is
very smooth and uniform (figure 2(A)) for 100% PPyNSE,
whereas the surface roughness increased with increasing
ratio of PPy (figures 2(B), (C) and (D)). This experimental
observation is in agreement with the already reported high
surface roughness for PPy coatings [20].

4.2. In situ copolymerization

During electrodeposition of monomers on a metal surface, the

bond strength between the metal surface and coating is an
important criterion for adhesion and stability of the coating.
Thus in this prospect, the average bond strength of different
copolymer coatings to the metal surface was determined using
a pull-off adhesion test. The maximum amount of pull-off
force applied with the hydraulic ram was measured and the
stress (N cm2) required to remove the coating (i.e. the nominal
coating adhesion strength) was determined and compared for
each copolymer proportion. It was found that as the proportion
of PPyNSE was increased in coating, it required more shear
stress (figure 3) to remove the coating from the steel surface.

PPy and PPyNSE were copolymerized on a metal surface by

electropolymerization of monomers in a non-aqueous solution.
The ratio of PyNSE to untreated Py was varied and its effect
on the surface properties, chemical composition and adhesion
of the coating to the metal surface was investigated. The
FTIR spectra of the coating material exhibited peaks at 1738.9
corresponding to the succinimidyl ester group and 1778.8
and 1811.9 cm1 for the pyrrolidinone group of succinimide
with other peaks at 1207 and 1068 cm1 for N-O and C-O
stretching, respectively. A peak at 1560 cm1 corresponding
238

4.3. Pull-off adhesion test

Immobilization of drugs and biomolecules on in situ copolymerized active ester polypyrrole coatings for biomedical applications

( A)

(B )

(C )

(D )

These results suggest that the PPyNSE attached coated surface

has more adherence strength as compared to PPy coated
surfaces and this coating may be suitable for the intended
use.
4.4. Immobilization of biomolecule (BSA)
The PPyNSEx coating with various fractions of NSE groups on
the surface was used for the attachment of proteins via covalent
bonds. The covalent attachment of BSA was determined by the
reactivity of active NSE groups with amino acid residues of the
bound BSA. The reactivity of the copolymer coating towards
BSA was examined by FTIR (figure 4) which explicitly
indicates the formation of an amide bond with reduction in
intensity of ester peak. Similarly, a BSA immobilization study
on different copolymer compositions was conducted and it
was found that the amount of BSA immobilized on the surface
increased (figure 5) with increasing PPyNSE fraction, thereby
enabling different levels of BSA loading. The PPyPPyNSE
coating thus can be used for the covalent attachment of proteins
with controlled surface proportion.

Force required (Newton/cm2 )

Figure 2. Scanning electron micrographs of the PPyNSE-PPy coated surfaces: (A) PPyNSE100, (B) PPyNSE75, (C) PPyNSE50 and (D) PPy.
The surface morphology of the coating indicates the formation of a smooth and uniform polymer coating on the metal surface by
PPyNSE100. Surface roughness increases as the ratio of pyrrole increases and the roughest surface was observed for PPy.

160

120

80

40

0
A

Diffrent coatings

Figure 3. Force required in a pull-off adhesion test to remove

different copolymers from 316L stainless steel plates:
(A) PPyNSE25, (B) PPyNSE50 and (C) PPyNSE100 (n = 3 for each
group).

4.5. Reactivity of active coating towards drugs

The PPyNSE coated surface was covalently attached with PNA
(a model molecule bearing an amino group) by incubating with
239

W Khan et al

Figure 4. FTIR of (A) freshly prepared PPyNSE coating and (B) BSA attached PPyNSE coating. It is clearly visible that the BSA attached
PPyNSE coating shows the presence of an amide peak and reduction in intensity of an ester peak.

PNA attached (mg/m2)

BSA attached (mg/cm2)

0.10
0.08
0.06
0.04
0.02

400
350
300
250
200
150
100
50
0
0.00

0.00
0

20

40

60

80

100

% PPyNSE

Figure 5. BSA attachment at different copolymer (PPyNSEPPy)

compositions: PPyNSE25, PPyNSE50, PPyNSE75 and
PPyNSE100 (n = 3 for each group). BSA attachment at different
copolymer compositions was carried out and it was clearly observed
that increasing the PPyNSE ratio in the copolymer composition
increases BSA attachment almost in a linear manner.

buffered PNA solution. For this purpose, PPyNSE coated

plates were incubated in different PNA concentrations and the
amount of covalently attached PNA was determined.
Figure 6 shows that the covalent attachment of PNA
increased with an increase in PNA concentration till the
saturation level which was achieved at about 0.2 mg ml1
of initial PNA concentration, and thereafter further increase
in concentration produced a negligible or no increase in PNA
attachment. Thus, it can be concluded that all surface reactive
groups have reacted with PNA molecules at this concentration,
and further increase in PNA concentration does not further
increase attachment. The dried coating was then characterized
by FTIR which showed a reduction in band intensities of NSE
and pyrrolidone groups due to the covalent attachment of PNA
molecules leading to the release of NSE groups. This is a
clear indication that NSE-functionalized coating is a useful
tool for covalent attachment of amino-containing molecules
on the surface.
240

0.20
0.40
PNA concentration (mg/ml)

0.60

Figure 6. Reactivity of PNA onto PPyNSE100 coated specimen (n =

3 for each group). By increasing PNA concentration, the covalent
attachment of drug increases to a saturation level. This saturation
was achieved at about 0.2 mg ml1 initial drug concentration;
thereafter, further increase in concentration produces less or no
increase in drug attachment.

5. Conclusion
Although PPy has been extensively employed over the past
few decades as a coating material, it is still associated
with certain limitations. Thus, there is an intense need
for modification/derivatization of PPy for improving the
processablity, functionality and physical properties of PPy
with other desired properties. With this background, Npyrrole derivative, i.e. active pyrrole ester, was synthesized
by activation of the carboxylic group with the N-succinimidyl
ester to form PyNSE. Then, in situ copolymerization of
synthesized monomer was carried out with Py. The ratio
of PyNSE to untreated Py was varied and the effect on
the surface morphology, chemical composition as well as
physical properties such as the adhesion strength of different
copolymer coatings on a metal surface were investigated.
The functionalized copolymer coating was then tested for
covalent attachment and its reactivity with BSA and PNA
via replacement of the NSE group, and was examined
by FTIR which unambiguously indicated the formation of
interfacial amide groups. Thus, one can conclude that

Immobilization of drugs and biomolecules on in situ copolymerized active ester polypyrrole coatings for biomedical applications

the covalent attachment of these molecules and controlled

surface proportion of the copolymer coating formed can be
used to express that functionalized coating is highly reactive
towards the amine-group-containing drugs and biomolecules
which make these surfaces suitable for various biomedical
applications.

Acknowledgments
WK is grateful to NIPER for a PhD fellowship. TM is also
grateful to the Ministry of Education, Ethiopian government
for a postgraduate scholarship. Financial support from
the Department of Science and Technology DST grant (#
SR/FTP/CSA-16/2003) to NK is duly acknowledged.

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