3350 Langmuir 2004, 20, 3350-3356

Synthesis and Characterization of Active

Ester-Functionalized Polypyrrole-Silica Nanoparticles:
Application to the Covalent Attachment of Proteins
Ammar Azioune,† Amel Ben Slimane,‡ Lobnat Ait Hamou,† Anne Pleuvy,†
Mohamed M. Chehimi,*,† Christian Perruchot,§ and Steven P. Armes§
ITODYS, Université Paris 7 - Denis Diderot, CNRS (UMR 7086), 1 rue Guy de la Brosse,
75005 Paris, France, Laboratoire de Chimie des Matériaux, Faculté des Sciences,
7021 Jarzouna, Tunisia, and Department of Chemistry, School of Life Sciences,
University of Sussex, Falmer, Brighton, BN1 9QJ United Kingdom

Received November 2, 2003. In Final Form: January 21, 2004

Novel ester-functionalized polypyrrole-silica nanocomposite particles were prepared by oxidative

copolymerization of pyrrole and N-succinimidyl ester pyrrole (50/50% initial concentrations), using FeCl3
in the presence of ultrafine silica nanoparticles (20 nm diameter). The N-succinimidyl ester pyrrole monomer
was prepared in aqueous solution using 1-(2-carboxyethylpyrrole) and N-hydroxysuccinimide in the presence
of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The resulting nanocomposites (N-succinimidyl ester
polypyrrole-silica) are raspberry-shaped agglomerates of silica sol particles “glued” together by the insoluble
poly(pyrrole-co-N-succinimidyl pyrrole). The N-succinimidyl ester polypyrrole-silica particles were
characterized in terms of their size, density, copolymer content, and polydispersity. Scanning electron
microscopy and disk centrifuge sedimentometry confirmed that the nanocomposite particles had narrow
size distributions. X-ray photoelectron spectroscopy analysis indicated a silica-rich surface and a high
surface concentration of N-succinimidyl ester groups. These nanoparticles exhibited good long-term
dispersion stability. The chemical stability of the ester functions in aqueous media after several weeks
of storage was monitored by FTIR spectroscopy. The functionalized nanocomposites were tested as
bioadsorbents of human serum albumin (HSA). The very high amount of immobilized HSA determined
by UV-visible spectroscopy is believed to be due to covalent binding. Incubation of the HSA-grafted
nanocomposite with anti-HSA resulted in immediate flocculation, an indication that they are alternative
candidates for visual diagnostic assays.

Introduction protein immobilization. However, the formation of a Schiff

Polypyrrole nanoparticles are a unique class of materials
with potential applications in visual immunodiagnostics
assays due to their intense optical absorbance.1 The facile
preparation of polypyrrole in aqueous media and its
surface modification by various specific functional groups
makes this polymer particularly suitable for the covalent
attachment of proteins. In this context, Tarcha et al.1
developed a method of surface functionalization of steri-
cally stabilized polypyrrole latex particles by the intro-
duction of chemical groups after N-acylation or C-acylation
using bromoacetyl bromide. The resulting latexes could
be further modified to bear carboxylic acid and amino
groups. This method, though rather complex, enabled the
covalent attachment of proteins and hence the develop-
ment of visual agglutination immunodiagnostic assays.
Miksa and Slomkowski2 prepared polypyrrole/polyacrolein
(PPy/PA) core-shell latex particles in order to introduce
surface aldehyde groups capable of reacting with proteins
via Schiff base formation. It is important to note that these
PPy/PA particles did not require any activation prior to
* Corresponding author. Fax: +33-144276814. E-mail: chehimi@
paris7.jussieu.fr.
† ITODYS, Université Paris 7 - Denis Diderot, CNRS (UMR
7086).
‡ Laboratoire de Chimie des Matériaux, Faculté des Sciences.
§ Department of Chemistry, School of Life Sciences, University
of Sussex.
(1) Tarcha, T.; Misun, D.; Finley, D.; Wong, W.; Donovan, J. J. Polymer
latexes: Preparation, characterization and application; Daniels, E. S.,
Sudol, E. D., El-Aassar, M. S., Eds.; ACS Symposium Series 492;
American Chemical Society: Washington, DC, 1992; p 347.
(2) Miksa, B.; Slomkowski, S. Colloid Polym. Sci. 1995, 47, 273.
base is reversible (at pH ≈ 9) and thus requires stabiliza-
tion by, for example, sodium cyanoborohydride.3
In the early 1990s, Armes and co-workers synthesized
a second class of colloidal polypyrrole particles by poly-
merizing pyrrole in the presence of ultrafine inorganic
particles such as silica.4 It was shown that these poly-
pyrrole-silica nanocomposites had a “raspberry” mor-
phology, with the silica particles being “glued” together
by the precipitating polypyrrole.5-7 Armes et al.5,6 suc-
cessfully synthesized carboxylic acid functionalized poly-
pyrrole-silica nanocomposites by copolymerizing a func-
tional pyrrolic comonomer (either 1-(2-carboxyethyl)pyrrole
or pyrrole-3-acetic acid, respectively) with pyrrole during
the nanocomposite synthesis. Amine-functionalized poly-
pyrrole-silica nanocomposites were synthesized via two
routes: (i) copolymerization of an N-substituted amino-
functional comonomer with pyrrole and (ii) the treatment
of nonfunctionalized nanocomposites with 3-amino-
propyltriethoxysilane (APTES). Since the latter nano-
composites had silica-rich surfaces, they readily reacted
with APTES to produce surface amine groups that were
grafted via silanol groups rather than pyrrole repeat
units.8
(3) Charleux, B.; Fanget, P.; Pichot, Makromol. Chem. 1992, 193,
205.
(4) Maeda, S.; Armes, S. P. J. Colloid Interface Sci. 1993, 159, 257.
(5) Maeda, S.; Corradi, R.; Armes, S. P. Macromolecules 1995, 28,
2905.
(6) McCarthy, G. P.; Armes, S. P.; Greaves, S. J.; Watts, J. F. Langmuir
1997, 13, 3686.
(7) Goller, M. I.; Barthet, C.; McCarthy, G. P.; Corradi, R.; Newby,
B. P.; Wilson, S. A.; Armes, S. P.; Luk, S. Y. Colloid Polym. Sci. 1998,
276, 1010.

10.1021/la030407s CCC: $27.50 © 2004 American Chemical Society

Published on Web 03/20/2004
Functionalized Polypyrrole-Silica Nanoparticles
The unfunctionalized and functionalized nanocompos-
ites (PPy-silica, PPy-silica-COOH, and PPy-silica-
NH2, respectively) were further tested as bioadsorbents
of DNA fragments9 and proteins.10 It was found that
unfunctionalized PPy-silica particles are much more
effective than the corresponding PPy bulk powders in
adsorbing human serum albumin (HSA) (147 and 63 mg/
g, respectively). In contrast, DNA adsorption was rather
weak onto the same PPy-silica particles at pH 7.4.9 In
this earlier work, it was found that immobilization of DNA
was only possible using surface-functionalized PPy-
silica-COOH and especially PPy-silica-NH2 particles.9
Pope et al.11 used N-carboxylic acid functionalized poly-
pyrrole-silica nanocomposites as marker particles in a
simple strip assay for the human pregnancy hormone,
hCG. However, covalent attachment of the monoclonal
anti-hCG required two surface treatment steps.
There are several ways to activate amino and carboxyl
groups for the effective immobilization of proteins to carrier
surfaces. The subject has been thoroughly described by
Gubitz in his review of the immobilization of proteins for
selective interaction with analytes in liquid chromatog-
raphy.12 For example, the use of some coupling agents
such as N-hydroxysuccinimide (NHS) in the presence of
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)
can activate the reaction between the primary amino
groups of the proteins and the esterified surfaces. Nikin
et al.13 applied this strategy to the immobilization of
the protein catalase onto carboxylic acid modified self-
assembled monolayers (SAMs) on gold substrates after
esterification by NHS in the presence of EDC. It was found
that immobilization resulted in increased protein adsorp-
tion. This method has attracted the attention of several
groups working on aqueous-phase peptide coupling14,15
and on biosensors.16,17
In this paper, we report the first example of surface
ester-functionalized polypyrrole-silica colloidal nano-
particles in aqueous media. Our strategy is based on
the synthesis of N-succinimidyl ester pyrrole monomer
(pyrrole-NHS) prior to copolymerization with pyrrole in
order to control the esterification of all carboxylic groups
present on the surface. The resulting nanocomposite
dispersion was characterized in terms of its particle size,
density, polypyrrole mass loading, surface chemical
composition (determined by X-ray photoelectron spec-
troscopy (XPS)), and long-term colloidal and chemical
stability. The nanocomposite particles were also tested as
a bioadsorbent of HSA from aqueous solution.
Experimental Section
1. Monomer Synthesis. 1-(2-Carboxyethyl)pyrrole was syn-
thesized according to the procedure described by Maeda et al.5
(8) Butterworth, M. D.; Corradi, R.; Johal, J.; Lascelles, S. F.; Maeda,
S.; Armes, S. P. J. Colloid Interface Sci. 1995, 174, 510.
(9) Saoudi, B.; Jammul, N.; Chehimi, M. M.; McCarthy, G. P.; Armes,
S. P. J. Colloid Interface Sci. 1997, 192, 269.
(10) Azioune, A.; Pech, K.; Saoudi, B.; Chehimi, M. M.; McCarthy,
G. P.; Armes, S. P. Synth. Met. 1999, 102, 1419.
(11) Pope, M. R.; Armes, S. P.; Tarcha, P. J. Bioconjugate Chem.
1996, 7, 436.
(12) Gübitz, G. In Selective Sample Handling and Detection in High-
Performance Liquid Chromatography; Frei, R. W., Zech, K., Eds.; Journal
of Chromatography Library, Vol. 39A, Part A; Elsevier: Amsterdam,
1988; pp 145-207.
(13) Nikin, P.; Martyn, C.; Davies, M.; Hartshorne, R. J.; Heaton, C.
J. R.; Saul, J. B. T.; Philip, M. W. Langmuir 1997, 13, 6485.
(14) Adamczyk, M.; Fishpaugh, J. R.; Mattingly, P. G. Tetrahedron
Lett. 1990, 40, 463.
(15) Corbett, A. D.; Gleason, J. L. Tetrahedron Lett. 2002, 43, 1369.
(16) Boukherroub, R. J.; Wojtyk, T. C.; Wayner, D. D. M.; Lockwood,
D. J. J. Electrochem. Soc. 2002, 149, H59.
(17) Wojtyk, J. T. C.; Morin, K. A.; Boukherroub, R.; Wayner, D. D.
M. Langmuir 2002, 18, 6081.
Langmuir, Vol. 20, No. 8, 2004 3351
1-(2-Cyanoethyl)-pyrrole (Aldrich) was hydrolyzed to 1-(2-car-
boxyethylpyrrole) by the addition of 11.92 mL of 1-(2-cyanoethyl)-
pyrrole to 60 mL of KOH (6.7 M) under helium. This mixture was
heated to reflux under an inert atmosphere until a red solution
was obtained. After 2 h, the disappearance of the ammonia was
confirmed using litmus paper. The solution was acidified to pH
5 using 8 M HCl at room temperature. The resulting product
was extracted five times with ether while maintaining the solution
at pH 5. After evaporation of the ether using a rotary evaporator,
the resulting oil became a beige solid after cooling overnight at
room temperature. The crude product was recrystallized from
boiling n-heptane. After removal of the solvent, white “needle”
crystals were formed, which were dried under a vacuum. The
structure of this product was confirmed by its melting point (66
°C) and NMR and IR spectra, which were in agreement with the
literature.
N-Succinimidyl ester pyrrole (Py-NHS) was synthesized as
follows: 0.863 g (7.5 mmol) of NHS (Acros) and 1.917 g (10 mmol)
of EDC (Sigma) were dissolved in 50 mL of distilled water, and
then 0.7 g (0.5 mmol) of 1-(2-carboxyethylpyrrole) was added to
the mixture. The reaction was carried out at ambient temperature
for 30 min. The white precipitate was collected by Büchner
filtration, washed with distilled water, and dried under a vacuum.
The melting point of the product was 159 °C, which was close to
that of NHS (150 °C).
The Py-NHS chemical structure has been characterized by
1H and 13C NMR spectroscopies using a Brücker AC200 spec-
trometer operating at 200.13 and 50.30 MHz, respectively. The
results are outlined below:
1H NMR (CDCl , ppm): 2.86 (s, 4H, CH CON); 3.08 (t, 2H, 7.2
3 2
Hz, CH2COO); 4.32 (t, 2H, 7.2 Hz, CH2N); 6.18 (dd, 2H, 2.2 and
2.2 Hz, CHR-pyrrole); 6.70 (dd, 2H, 2.2 and 2.2 Hz, CHβ-pyrrole).
13C NMR (CDCl , ppm): 25.8 (2 × CH CON); 33.5 (CH COO);
3 2 2
44.1 (CH2N); 108.9 (CHβ-pyrrole); 120.4 (CHR-pyrrole); 166.2 (CO2);
168.7 (2 × CON).
2. Synthesis of N-Succinimidyl Ester Polypyrrole-Silica
Nanocomposite Particles (Polypyrrole-Silica-NHS). The
N-succinimidyl ester polypyrrole-silica nanoparticles were
prepared according to the procedure described by Maeda et al.5
for the preparation of carboxylated nanocomposites. A typical
preparation was carried out as follows: 9.50 mL of an aqueous
solution of colloidal silica sol (30% w/w, Ludox products; 20 nm
diameter) was added to 28 mL of distilled water. Then, 0.25 mL
(3.58 mmol) of purified pyrrole (passed through a silica column
prior to use) and 0.845 g (3.58 mmol) of Py-NHS monomer were
added to the silica suspension. A solution of FeCl3‚6H2O (4.55
g) in 12.5 mL of deionized water was then added to this mixture,
and the polymerization was allowed to proceed overnight. The
final product was centrifuged several times at 10 000 rpm for 30
min and sonicated in order to purify the nanocomposite and to
eliminate the excess of silica sol. The purity of the nanocomposite
was checked by monitoring the solution pH of the final wash (pH
5.6). The nanocomposite particles were then redispersed in
deionized water with the aid of an ultrasonic bath. The final
nanocomposite dispersions are highly stable in water and do not
suffer any flocculation for more than 1 year.
3. Protein Immobilization. Human serum albumin (Sigma
product, fraction Cohen V) was immobilized onto polypyrrole-
silica-NHS nanoparticles in 0.1 M PBS at pH 7.4 according to
the protocol previously described by Azioune et al.18 Briefly, 400-
500 µL (corresponding to 10 mg of solid) of colloidal solution was
conditioned in 9.5 mL of PBS overnight to allow ion exchange to
occur.9,18 The suspension was then centrifuged to separate the
as-conditioned solid particles. These were further immersed in
10 mL of PBS containing different HSA concentrations. The “one-
shot” method was applied with an initial protein concentration
varying from 0 to 500 µg/mL. Incubation of HSA with colloidal
suspension was carried out overnight at room temperature in
Pyrex glass tubes with gentle stirring using a Speci-Mix
apparatus. After incubation, the suspension was centrifuged for
the second time, and the nonadsorbed HSA concentration
remaining in the supernatant was determined by the Bradford
method,19 using UV-visible spectroscopy. The amount of
(18) Azioune, A.; Chehimi, M. M.; Miksa, B.; Basinska, T.; Slomkow-
ski, S. Langmuir 2002, 18, 1150.
3352 Langmuir, Vol. 20, No. 8, 2004
Figure 1. Schematic representation of the synthesis of (a) N-succinimidyl ester pyrrole (pyrrole-NHS) and (b) the N-succinimidyl
ester polypyrrole-silica nanoparticles by copolymerization of pyrrole-NHS with pyrrole in the presence of ultrafine silica using
FeCl3 as the oxidizing agent.
immobilized protein was calculated by the depletion method using
M ) (Ci - C0)Vsol/msolid
where M is the amount of immobilized HSA (mg/g) and Ci and
C0 are the initial and equilibrium HSA concentrations (mg/mL),
respectively. Vsol is the total volume solution (mL), and msolid is
the amount of the polypyrrole-silica nanocomposite (g). The solid
was washed three times with 0.02% Tween 20 in PBS in order
to remove the physisorbed protein and twice with PBS, followed
by one wash with pure water (MilliQ). The solid was then dried
under a vacuum before XPS analysis.
4. Analytical Techniques. 4.1. Chemical Composition.
The chemical compositions of the polypyrrole-silica-NHS
particles were obtained from thermogravimetric analyses (Per-
kin-Elmer TGA-7 instrument; scan rate, 40 °C/min in air). Each
nanocomposite was heated to 800 °C, and the observed weight
loss was attributed to the quantitative pyrolysis of the organic
component. FTIR spectra (KBr disk) were obtained for the dried
particles using a a Nicolet Magna 550 Series II instrument
equipped with an MCT detector. Typically 100 scans per spectrum
were recorded at 4 cm-1 resolution.
4.2. Particle Size and Colloidal Stability. Disk centrifuge
photosedimentometry (DCP) was used to determine the mean
particle diameters of the nanocomposite particles. The analyses
were carried out using a Brookhaven instrument at 25 °C,
according to the protocol described by McCarthy et al.6 The
weight-average particle diameter and standard deviations were
calculated assuming normal statistics for the size distributions.
The particle density required for DCP analyses was determined
using a Micrometrics Acc-Pyc 1330 helium pycnometer. Each
density value was the average of three measurements.
4.3. Surface Analysis by XPS. The surface compositions of
the N-succinimidyl ester pyrrole monomer and its conjugated
copolymer nanocomposite were examined by XPS using a Surface
Science Instruments spectrometer (SSX-100 model). The dried
samples were mounted on a powder sample holder. The base
pressure during analysis was typically 5 × 10-9 Torr. The
monochromatic Al KR X-ray source (1486.6 eV) was used at 10
kV and 20 (or 12) mA. The X-ray spot size was 1000 and 600 µm
for the acquisition of the survey and narrow scan regions,
respectively. The pass energy in the hemispherical analyzer was
set at 150 eV for the survey spectra and 50 eV for the high-
(19) Bradford, M. M. Anal. Biochem. 1976, 72, 248.
Azioune et al.
resolution spectra. Under these conditions, the full width at half-
maximum (fwhm) of the C1s peak of the polypyrrole-silica-
NHS nanocomposite was 2.0 eV (for comparison, the SSI machine
manufacturer ensures an fwhm of 1.17 eV for Au4f7/2 from a
clean gold surface). The takeoff angle analysis relative to the
surface was 35°. A flood gun was used in order to minimize the
static charging effect. Peak fitting was carried out using the
Winspec software, kindly supplied by the LISE Laboratory at
University of Namur (Belgium). The binding energy scale was
calibrated by setting the main component due to the C-C/C-H
carbon type bonds at 284.6 eV. Both linear and Shirley
backgrounds were used, depending on the shape of spectra. The
surface atomic composition was calculated by the integration of
the peak areas on the basis of the elemental sensitivity factors
provided by the manufacturer.
Results and Discussion
1. Strategy for the Synthesis of the Py-NHS
Monomer and Polypyrrole-Silica-NHS Nano-
composite. Figure 1 depicts the synthesis of the Py-
NHS and its addition to the reaction medium to prepare
the polypyrrole-silica-NHS nanocomposite. The choice
of a 50:50 feed ratio for the copolymerization was suggested
by the work of McCarthy et al.6 who copolymerized pyrrole-
3-acetic acid with pyrrole. This feed ratio gave nanopar-
ticles that possessed a narrow size distribution and a
reasonable degree of surface carboxylation. More impor-
tantly, just a few surface chemical groups might be reactive
toward the functional groups of the immobilized proteins
due to the large size of these macromolecules.20
Before describing the characterization of the nanocom-
posite particles in detail, we shall first report the FTIR
and XPS results obtained with the Py-NHS monomer.
Its FTIR spectrum (see Figure 2, solid line) showed no
evidence for any band at 1706 cm-1 due to the carboxylic
groups of the 1-(2-carboxyethyl)pyrrole monomer, where-
as several new peaks at 1738, 1781, and 1816 cm-1
corresponding to the succinimidyl ester group and the
(20) Zammatteo, N. Ph.D. Thesis, University of Namur, Namur,
Belgium, 1998.
Functionalized Polypyrrole-Silica Nanoparticles Langmuir, Vol. 20, No. 8, 2004 3353

Figure 2. FTIR spectra of the 1-(2-carboxyethylpyrrole) before

(dashed line) and after esterification (solid line).

pyrrolidinone group of succinimide were prominent.21 In

addition, N-O and C-O stretches are evident at 1206
and 1067 cm-1, respectively.16
XPS analysis of the pyrrole-NHS monomer was con-
ducted since this was a useful reference compound for the
interpretation of the nanocomposite spectra. Figure 3
shows the survey scan and the C1s and O1s envelopes for
this monomer. The survey scan exhibits C1s, N1s, and
O1s peaks centered at ca. 285, 400, and 532 eV, respec-
tively. There is also a small degree of contamination by
silicon in the form of silica leached from the glass vessel
(difficult to remove) used for the monomer synthesis (Si2p
and Si2s centered at 103 and 151 eV, respectively).
The C1s spectra of both the Py-NHS (Figure 3b) and
the corresponding nanocomposite (discussed below) were
peak-fitted with four components (Table 1). At the high
binding energy side, a fairly intense component centered
at 288.1 eV was observed, which is characteristic of an
amide-type carbon. This signal is assigned to the N-CdO
(pyrrolidinone) function of the succinimide group. One
can also observe a less intense high binding energy
component at 289.3 eV, which is characteristic of the ester
carbon of the pyrrole-NHS.
From Table 1, the N-CdO/O-CdO ratio is approxi-
mately 2, which is in good agreement with the chemical
structure of this monomer (see Figure 1). The pyrrole-
NHS O1s peak (Figure 3c) was peak-fitted with two
components centered at 532 and 534 eV. These components
Figure 3. XPS spectra of the N-succinimidyl ester pyrrole: (a)
can be assigned to the carbonyl groups and the C-O-N survey scan and (b) C1s and (c) O1s regions.
oxygen atom from the pendent succinimide group. The
CdO/C-O-N relative intensity is 3, as expected for the Table 1. Peak Fitting Parameters for the C1s Core Level
monomer structure. from the N-Succinimidyl Ester Pyrrole and
2. Synthesis and Properties of the Polypyrrole- N-Succinimidyl Ester Functionalized Polypyrrole-Silica
Silica-NHS Nanocomposite. The novel N-ester-func- Nanocomposite
tionalized polypyrrole-silica nanocomposite particles Py-NHS PPy-NHS-silica
have the same intense black color as the unfunctionalized C-C/C-Hx (284.6 eV) 59.8 59.2
homopolypyrrole-silica nanocomposites. This is charac- C-N (286 eV) 21.8 24.4
teristic of the highly conjugated structure of polypyrrole N-C ) O (288.1 eV) 12.2 11.1
and, in the context of visual agglutination assays, makes N-O-(C)O)-C (289.3 eV) 6.2 5.3
these intrinsically chromogenic particles an interesting
alternative to conventional extrinsically dyed polystyrene in fair agreement with the scanning electron microscopy
latex. The polypyrrole-silica-NHS nanoparticles have (SEM) pictures (see Figure 4), indicating an average size
good colloidal stability, as judged from DCP measure- of 175 ( 25 nm (average values calculated for 10 particles).
ments. Density measurements by helium pycnometry gave a value
The weight-average particle diameter (Dw) obtained of 1.697 g cm-3 for the functionalized nanocomposites.
by DCP was 210 ( 36 nm. Polydispersities are given by Assuming the densities of 2.16 and 1.5 for silica and
the ratio of Dw to the number-average diameter (Dn) and N-succinimidyl polypyrrole (PPy-NHS), it follows that
clearly show that these colloidal particles have relatively the weight fraction of silica is ca. 30%. [The assumed
narrow size distributions. DCP results were found to be density of PPy-NHS is based on the values of 1.53 and
1.45 for polypyrrole and poly(pyrrole-3-acetic acid),6
(21) Bäuerle, P.; Hiller, M.; Scheib, S.; Sokolowski, M.; Umbach, E. respectively. Since the pendent NHS group is much larger
Adv. Mater. 1996, 3, 8. than the COOH, it is in principle expected that pure PPy-
3354 Langmuir, Vol. 20, No. 8, 2004 Azioune et al.

Figure 4. Scanning electron micrographs of the polypyrrole-

silica-NHS particles.

Figure 6. XPS spectra of the polypyrrole-silica-NHS nano-

composite: (a) survey scan and (b) C1s high-resolution spec-
trum.

Table 2. Surface Atomic Compositions of

Polypyrrole-Silica Nanocomposites after the
Immobilization of HSA in 0.10 M PBS at pH 7.4
materialsa C O N Si Na
PPy-silica-NHS 37.8 42.5 5.8 13.8 0
Figure 5. FTIR spectrum of the polypyrrole-silica-NHS PPy-silica-NHS-HSA-10 µg/mL 50 39.5 4.2 5.4 0.7
nanocomposite after 4 months of storage as an aqueous PPy-silica-NHS-HSA-20 µg/mL 56.8 32.3 5.4 4.6 0.9
suspension at pH ∼ 2-3. PPy-silica-NHS-HSA-50 µg/mL 64.2 24.3 6.3 4 1.2
PPy-silica-NHS-HSA-150 µg/mL 58.3 27.5 7.7 4.6 1.5
NHS (100% Py-NHS repeat units) would have a lower PPy-silica-NHS-HSA-300 µg/mL 69 20.3 6.4 3.7 1.3
density than 1.45. However, since we are dealing with a PPy-silica-NHS-HSA-500 µg/mL 60 25.6 7.1 4.4 3
copolymer of pyrrole and Py-NHS, it follows that the a x µg/mL stands for the initial concentration of HSA used for

density of the conducting polymer is between 1.4 and 1.53,
hence the assumed value of 1.5.] Thermogravimetric
analyses gave a value of 62% w/w PPy-NHS, however
uncorrected for the moisture content of silica. Taking into
account the 12% surface moisture content of the silica sol,
it follows that the true PPy-NHS mass loading is ca. 65%
w/w, which is in reasonable agreement with the mass
loading of 59% indicated by microanalytical data.
The nanocomposite density can therefore be corrected
to the value of 1.735 g cm-3 (instead of 1.697 g cm-3)
assuming the 35 and 65% w/w of silica and the copolymer,
respectively, in the dry nanocomposite.
FTIR spectroscopy was employed to characterize the
Py-NHS repeat units within the nanocomposite. For the
nanocomposite, Figure 5 exhibits a broad peak at 1117
cm-1 characteristic of silica which is the superimposition
of three Si-O stretching vibrations6 and confirms that
the ester group is indeed detected by its corresponding
peak centered at 1738 cm-1. This specific feature of the
functionalized PPy does not appear in the spectrum of the
unmodified silica. More importantly, the peak at 1706-
1709 cm-1 characteristic of the carboxyl group does not
appear. This is a clear indication of the stability of the
ester group. Both the monomers of the Py-COOH, the
determining the attachment isotherm.
Py-NHS, and the nanocomposite have been analyzed by
FTIR in the same conditions (i.e., pH ∼ 2-3). Taking into
account the acidic pH of the suspension and the KBr disks
of the dried nanocomposites, it is unlikely to have the
anionic carboxylate form of the hydrolyzed nanocomposites
as was shown with the Py-COOH monomer (pH ∼ 2-3).
Therefore, FTIR brings strong supporting evidence for
the stability of the NHS groups at the surface of the
nanocomposites, even after a prolonged period of 4 months.
Figure 6 displays both the XPS survey and high-
resolution spectra obtained for the nanocomposite par-
ticles. The survey scan (Figure 6a) is similar to that of
pure silica with the addition of low-intensity C1s and N1s
signals. This indicates that these functionalized nano-
composite particles, like the unfunctionalized homopoly-
pyrrole-silica nanocomposites reported earlier,22 have
silica-rich surfaces (see below, Table 2). Chlorine should
be detected, since this is the dopant counterion, but
because there is only one chloride anion per three or four
(22) Maeda, S.; Gill, M.; Armes, S. P.; Fletcher, I. W. Langmuir 1995,
11, 1899.
Functionalized Polypyrrole-Silica Nanoparticles Langmuir, Vol. 20, No. 8, 2004 3355

Figure 7. Schematic illustration of the chemical reaction

between the polypyrrole-silica-NHS nanocomposites and the
pendent amine groups of a protein resulting in covalent
attachment via amide formation.

pyrrole units, it is near the detection limit. The C1s

spectrum (Figure 6b) has a similar shape to that of the
Py-NHS monomer and has been fitted with four com-
ponents; the peak fitting parameters are reported in Table
1. A high binding energy component centered at 288.1 eV
can be assigned to the amide groups (N-CdO) from the
NHS pendent groups. This component seems to be very
stable, and no evidence was found for any hydrolysis on
long-term storage in aqueous solution.
The relative intensities of the carbon atom that is
characteristic of the succinimide (288.1 eV) and that of
the formed ester (289.4 eV) are in agreement with those
obtained for the Py-NHS monomer.
Combining the surface composition in atomic % in Table
2 and the results reported in Table 1 for the C1s peak
fitting, the surface composition of the nanocomposite,
within the analysis volume dv probed by XPS, is estimated
to be approximately 78% silica, 13 ( 2% pyrrole, and 9 (
2% Py-NHS, respectively. [This calculation was based on
the estimation of the ester function from the relative
intensity of the C1s component. By multiplying this
Figure 8. XPS survey spectra of the polypyrrole-silica-NHS
proportion by 11 (the number of carbon atoms in the Py- nanocomposite after incubation with human serum albumin at
NHS repeat units) and subtracting from the total C1s an initial concentration of (a) 50 µg/mL and (b) 500 µg/mL.
area, one obtains the contribution of pyrrole and pyrrole-
NHS repeat units to the total C1s peak area from the for nonporous particles, one can estimate the surface area
copolymer in the nanocomposite.] Thus, quantitatively, (A):
the nanocomposites have a silica-rich surface, on one hand,
and the relative concentrations of pyrrole and Py-NHS A ) 3/(Fr)
at the particle surface correspond approximately to the
initial comonomer feed prior to copolymerization, on the where F is the density (1.735 g cm-3) and r is the radius
other hand. (75-100 nm) of the nanocomposites. The calculated value
3. Immobilization of Human Serum Albumin onto is ca. 20 ( 3 m2/g. However, Maeda and Armes4 have
Polypyrrole-Silica-NHS Nanocomposites. The poly- shown that a correction factor of at least 2.0 must be
pyrrole-silica-NHS nanocomposite was evaluated as a introduced in order to take into account the microporosity
bioadsorbent of a model protein, HSA. The chemical and roughness of the particles. We therefore estimate the
reaction leading to protein immobilization on the nano- specific surface area to be 40-50 m2/g. This value yields
composite is depicted in Figure 7. The NHS group reacts an immobilization of protein estimated to be 6-7.5 mg/
with the protein, and an amide bond is formed. The m2, which is in agreement with published data on
maximum amount of immobilized HSA determined using monolayer adsorption of HSA with end-on conformation.23
the Bradford method was approximately 300 mg g-1 for The covalent attachment of the protein onto active ester-
an initial protein concentration of 500 µg mL-1. This is a functionalized polypyrrole-silica nanoparticles is due to
much higher loading than those found in our previous the chemical reaction between amino groups of the protein
studies with either unfunctionalized polypyrrole-silica and the pendent ester groups from the functionalized
nanocomposite10 or powders.18 The N-succinimidyl ester pyrrole units (see Figure 7). This is very likely and
functionalization of the polypyrrole-silica nanocomposites supported by the work of Korri-Youssoufi et al.24 These
is thus a prequisite for an effective and massive attach- authors reported FTIR results on the immobilization of
ment of proteins. the oligonucleotide (ODN) onto 3-functionalized poly-
In addition to the presence of surface reactive groups, pyrrole with ester groups that are in line with a covalent
the high specific surface area of the nanocomposite also bond formation between the conducting substrate and the
plays an important role, and that is the reason adsorption biological macromolecule. Indeed, during ODN attach-
is very high in milligrams of protein per gram of colloid. ment, they observed a disappearance of the frequencies
As outlined below, the adsorption in mg/m2 units remains, associated with pyrrolidinedione at 1820 and 1786 cm-1
however, in line with the range of adsorption of proteins and the appearance of the frequency corresponding to the
onto colloidal particles. Indeed, the nanocomposites have
a raspberry shape that should result in a high specific (23) Douglass, R. J.; Sasha, O.; Sharon, G. R. Langmuir 2000, 16,
5449.
surface area as for the unfunctionalized PPy-silica (24) Kourri-Youssoufi, H.; Makrouf, B. Anal. Chim. Acta 2002, 85,
nanoparticles.4 Using the following equation established 469.
3356 Langmuir, Vol. 20, No. 8, 2004
Figure 9. Plot of N/Si atomic ratio for the polypyrrole-silica-
NHS nanocomposite versus the initial concentration of HSA
used for the incubation of the particles.
amide group at 1715 cm-1. In the present work, it was
difficult to follow the reaction from the pyrrolidinedione
group because the relative intensities are too weak even
for the “virgin” polypyrrole-silica-NHS nanoparticles
(see Figure 5).
After incubation with HSA, the nanocomposites were
characterized by XPS. Figure 8 shows the survey spectra
of the nanocomposite for two different initial HSA
concentrations. There is a substantial modification in the
structure of the survey scan after adsorption of HSA in
comparison to the spectrum of the untreated nanocom-
posite (Figure 6a). The immobilization of the protein is
indicated by the relative increase in the C1s and N1s peak
intensities. At the same time, a significant decrease in
the Si2p peak intensity is observed. Also, sodium was
detected by its Na1s and NaKLL peaks (centered at 1072
and 493 eV, respectively), whose relative intensities
increase for higher HSA concentrations. The presence of
sodium at the nanocomposite-HSA interface is most
probably due to the charge neutralization of negatively
charged residues from the protein.
Table 2 reports the surface compositions of the nano-
composites after HSA immobilization at the stated initial
concentrations. The reduction in the silicon content occurs
at low amounts of immobilized protein, suggesting that
the surface reached saturation for relatively low initial
HSA concentrations. Indeed, plotting surface N/Si atomic
ratio versus the initial HSA concentration (Figure 9) yields
a high affinity type isotherm for protein immobilization.
Azioune et al.
A steady state is obtained for initial HSA concentration
higher than 50 µg/mL.
To perform a qualitative (Yes/No) visual agglutination
test, we incubated the HSA-decorated nanocomposite
particles with anti-HSA at an initial concentration of 200
µg mL-1 (added volume, 100 µL of anti-HSA solution). A
bridging flocculation readily took place due to multiple
antibody-antigene specific reactions between anti-HSA
(antibody) and the surrounding nanocomposite-immobi-
lized HSA (antigene). The same test performed using a
200 µg mL-1 HSA solution (100 µL volume) added to a
suspension of HSA-decorated nanocomposite particles was
negative since the nanoparticles remained dispersed.
Conclusions
Novel polypyrrole-silica nanocomposite particles bear-
ing reactive surface N-hydroxysuccinimide functional
groups were prepared in aqueous solution by copoly-
merization of pyrrole and N-esterified pyrrole (pyrrole-
NHS) using FeCl3 in the presence of an ultrafine silica
sol. Both disk centrifuge photosedimentometry and SEM
showed that the nanocomposite particles have a mean
diameter around 200 nm and a reasonably narrow size
distribution. XPS and FTIR spectroscopy confirmed the
existence and the chemical stability of the desired ester
group at the surface of the colloid particles. The molar
ratio of pyrrole-NHS/pyrrole repeat units is in the 1:1 to
1:2 range for an initial 1:1 comonomer feed. These
functionalized particles proved to be effective for the
covalent immobilization of a model protein, namely, HSA.
Moreover, the HSA-coated nanocomposites readily reacted
with anti-HSA with a result of flocculation.
The physicochemical characteristics of the novel surface-
functionalized polypyrrole-silica nanocomposites, to-
gether with their excellent chemical and colloidal stability,
provide a very interesting alternative to previously
evaluated polypyrrole-based particles and conventional
dyed polystyrene latexes for biomedical applications such
as visual agglutination immunodiagnostic assays and
nanoparticle-based biosensors.
Acknowledgment. The authors thank Mrs. M.-J.
Vaulay for the SEM images and Dr. G. Trippé for her
assistance with NMR spectral interpretation. Dr. A.
Adenier and Mr. S. Bousalem are acknowledged for their
assistance with FTIR. A. Ben Slimane thanks the Uni-
versity Paris 7 - Denis Diderot for financial support
through an invited professorship.
LA030407S