10940
Langmuir 2007, 23, 10940-10949
Magnetic Fe2O3-Polystyrene/PPy Core/Shell Particles: Bioreactivity and Self-Assembly Claire Mangeney,*,† Meriem Fertani,† Smain Bousalem,‡ Ma Zhicai,† Souad Ammar,† Fre´deric Herbst,† Patricia Beaunier,§ Abdelhamid Elaissari,| and Mohamed M. Chehimi† Interfaces, Traitements, Organisation et Dynamique des Syste` mes (ITODYS), UniVersite´ Paris 7 Denis Diderot, CNRS (UMR 7086), 1 rue Guy de la Brosse, 75005 Paris (France), Centre UniVersitaire Mustapha Stombouli de Mascara, Institut des Sciences et de Technonologies, BP 763, 29000 Mascara, Alge´ rie, Laboratoire de Re´ actiVite´ des Surfaces, UMR-CNRS 7609, UniVersite´ Pierre et Marie Curie, 4, place Jussieu 75251 Paris, France, and Unite Mixte CNRS-BioMe´ rieux, ENS de Lyon, 46, Alle´ e d’Italie, 69364 Lyon, France ReceiVed February 19, 2007. In Final Form: July 14, 2007 This paper describes the synthesis of new magnetic, reactive polystyrene/polypyrrole core/shell latex particles. The core consists of a polystyrene microsphere containing γ-Fe2O3 superparamagnetic nanoparticles (PSmag), and the shell is made of reactive N-carboxylic acid-functionalized polypyrrole (PPyCOOH). These PSmag-PPyCOOH latex particles, average diameter 220 nm, were prepared by copolymerization of pyrrole (Py) and the active carboxyl-functionalized pyrrole (PyCOOH) in the presence of PSmag particles. PNVP was used as a steric stabilizer. The functionalized polypyrrole-coated PSmag particles were characterized in terms of their particle size, surface morphology, chemical composition, and electrochemical and magnetic properties using transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), cyclic voltammetry, and SQUID magnetometry. Activation of the particle surface carboxyl groups was achieved using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS), which helps transform the carboxyl groups into activated ester groups (NSE). The activated particles, PSmagPPyNSE, were further evaluated as bioadsorbents of biotin used as a model biomolecule. It was shown that biotin was immobilized at the surface of the PSmag-PPyNSE particles by forming interfacial amide groups. The assemblies of PSmag-PPyCOOH particles on glass plates were further investigated. When no magnetic field is applied, the particles assemble into 3D colloidal crystals. In contrast, under a magnetic field, one-particle-thick chains gathered in hedgehog-like architectures are obtained. Furthermore, PSmag-PPyCOOH coated ITO electrodes were shown to be electroactive and electrochemically stable, thus offering potentialities for creating novel high-specific-area materials for biosensing devices where the conducting polymer component would act as the transducer through its conductive properties.
1. Introduction Colloidal particles with controlled core-shell morphology have increasingly attracted interest due to their potential applications in photonic crystals, multi-enzyme biocatalysis, and drug delivery systems.1 Particularly, several publications have reported on the synthesis of polystyrene cores (PS) coated with inherently conducting polymers (ICPs)2 such as polypyrrole3 (PPy), polyaniline4 (PAni), and polyethylenedioxythiophene5 (PEDOT). This colloidal approach was extensively studied not only because it allows to improve the processability of conducting polymer materials, but also because of the attractive properties of these materials such as well-defined colloidal dimensions or morphologies,2b intense coloration, biocompatibility,6 high surface area, efficient radiation absorption, wide spectrum of surface * Corresponding author: Dr. Claire Mangeney, mangeney@ paris7.jussieu.fr, Tel: +33 1 4427 6834, Fax: +33 1 4427 6814. † CNRS (UMR 7086). ‡ Institut des Sciences et de Technonologies. § UMR-CNRS 7609. | Unite Mixte CNRS-BioMe ´ rieux. (1) Caruso, F. AdV. Mater. 2001, 13, 11. (2) (a) Armes, S. P. In Handbook of Conducting Polymers, 2nd ed.; Skotheim, T. A., Elsenbaumer, R. L., Reynolds, J. R., Eds.; Marcel Dekker: New York, 1998; Chapter 17, p 423. (b) Khan, A.; Armes, S. P. AdV. Mater. 2000, 12, 671. (3) Cairns, D. B.; Armes, S. P.; Chehimi, M. M.; Perruchot, C.; Delamar, M. Langmuir 1999, 15, 8059. (4) Barthet, C.; Armes, S. P.; Lascelles, S. F.; Luk, S. Y.; Stanley, H. M. E. Langmuir 1998, 14, 2032. (5) Han, M. G.; Armes, S. P. Langmuir 2003, 19, 4523. (6) Li, Y.; Neoh, K. G.; Kang, E.-T. J. Colloid Interface Sci. 2004, 275, 488.
functionalization,7-12 good film-forming properties,13 and so forth. Specific applications that have been suggested include antistatic coatings,14 corrosion control of non-noble metals,14,15 calibration of the Cassini cosmic dust analyzer using polypyrrole-coated PS particles as analogues of carbonaceous micrometeoroids,16 and visual immunodiagnostic assays.17,18 For this latter application, the deep black color of polypyrrole particles, rather than their electrical conductivity, was the “added-value” property on which the agglutination assays were based. (7) Butterworth, M. D.; Corradi, R.; Johal, J.; Lascelles, S. F.; Maeda, S.; Armes, S. P. J. Colloid Interface Sci. 1995, 174, 510. (8) Slomkowski, S. Prog. Polym. Sci. 1998, 23, 815. (9) Azioune, A.; Ben Slimane, A.; Ait Hamou, L.; Pleuvy, A.; Chehimi, M. M.; Perruchot, C.; Armes, S. P. Langmuir 2004, 20, 3350. (10) . Mangeney, C.; Bousalem, S.; Connan, C.; Adenier, A.; Baunier, P.; Chehimi, M. M. Langmuir 2006, 22, 10163. (11) . Yip, Y.; Benabderrahmane, S.; Zhicai, M.; Bousalem, S.; Mangeney, C.; Chehimi, M. M. Surf. Interface Anal. 2006, 38, 535. (12) Boukerma, K.; Micˇusˇ´ık, M.; Mravcˇa´kova´, M.; Omastova´, M.; Vaulay, M.-J.; Beaunier, P.; Chehimi, M. M. Colloids Surf., A 2007, 293, 28. (13) Wiersma, A. E.; vd Steed, L. M. A.; Jongeling, T. J. M. Synth. Met. 1995, 71, 2269. (14) Wiersma, A. E.; vd Steed, L. M. A. Eur. Patent No. 1993, 589, 529. (15) Abu, Y. M.; Aoki, K. J. Electroanal. Chem. 2005, 583, 133. (16) Goldsworthy, B. J.; Burchell, M. J.; Cole, M. J.; Green, S. F.; Leese, M. R.; McBriders, N.; McDonnell, J. A. M.; Mtiller, M.; Grtin, E.; Srama, R.; Armes, S. P.; Khan, M. A. AdV. Space Res. 2002, 29, 1139. (17) Tarcha, P. J.; Misun, D.; Finley, D.; Wong, M.; Donovan J. J. In Polymer Latexes: Preparation, Characterization and Applications; Daniels, E. S., Sudol, E. D., El-Aassar, M. S., Eds.; ACS Symposium Series No. 492; American Chemical Society: Washington, DC, 1992; Vol. 22, p 347. (18) Bousalem, S.; Benabderrahmane, S.; Yip Cheung Sang, Y.; Mangeney, C.; Chehimi, M. M. J. Mater. Chem. 2005, 15, 3109.
10.1021/la700492s CCC: $37.00 © 2007 American Chemical Society Published on Web 09/28/2007
Fe2O3-PS/PPy Core/Shell Particles
In order to take advantage of the conductive properties of the polypyrrole-coated latexes and to improve by that way the accuracy of the biosensing event, it would be interesting to prepare conducting latex particle assemblies on planar surfaces or electrodes. Indeed, it would afford an electrode with, at the same time, an enhanced specific area and an electroactive layer able to transduce the biorecognition event. However, despite a massive amount of work on the synthesis, characterization and use of colloidal particles coated with conducting polymer shells in solution, assembling those latex particles onto planar substrates has proven to be challenging. Indeed, contrary to uncoated polystyrene latex particles, for which several assembly methods have been reported, PPy latex particles possess a high surface energy leading to aggregation of the particles. For instance, Armes et al.19 attached sterically stabilized polypyrrole latex particles onto gold substrates and studied the interaction of these modified surfaces with imidazole and L-histidine. The surface-anchored polypyrrole latex particles were found to form a discontinuous particles film. More recently, Yip et al.11 obtained 2D hexagonal assemblies from PPyCOOHcoated PS particles. However, the 2D particle layer was rather heterogeneous with the presence of many defects. In this work, we propose to study the assembling properties of polypyrrole-coated latex particles, under a magnetic field. In order to obtain particles that respond to a magnetic field, latex particles containing magnetic nanoparticles were used. Efforts have already been directed toward incorporating magnetic particles into core-shell structures of PPy. For instance, coreshell Fe3O4-polypyrrole nanoparticles were synthesized by direct polymerization of pyrrole in the presence of Fe3O4 nanoparticles.20 The thermal stability of Fe3O4-polypyrrole nanocomposites increased owing to interactions between Fe3O4 particles and polypyrrole chains, but the particles, with size ranging from 30 to 40 nm, were highly polydisperse. Other magnetic PPy colloids were prepared by deposition of silica onto ultrafine magnetite particles, followed by chemical polymerization of pyrrole.21 However, electron microscopy studies indicated a rather nonhomogenous core-shell morphology. In the present work, a new type of polypyrrole-coated magnetic core-shell particle was synthesized, which consists of γ-Fe2O3 superparamagnetic nanoparticle-containing polystyrene cores coated by a functionalized polypyrrole shell. The polystyrene particles provide a rigid, nondeformable, and nonporous support for PPy coating. We describe the preparation, characterization, and reactivity study of these polypyrrole-coated magnetic polystyrene latex particles bearing surface carboxyl groups. The magnetic polystyrene latex particles PSmag, diameter 190 nm, are made of a core of Fe2O3 nanoparticles covered with a layer of polystyrene (see Figure 1). The synthesis of these core-shell magnetic latexes was achieved by transforming oil-in-water magnetic emulsion into magnetic latex particles.22 The reactive conducting polymer coatings consist of copolymers of pyrrole and N-alkyl-substituted pyrrole with N-carboxyl groups (pyrroleCOOH) at the alkyl chain end in the initial 50/50 (%) ratio. Poly(N-vinylpyrrolidone), PNVP, was used as a steric stabilizer. The PSmag-PPyCOOH particles were characterized by means of transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), (19) Bjorklund, R. B.; Armes, S. P.; Maeda, S.; Luk, S. Y. J. Colloid Interface Sci. 1998, 197, 179. (20) Deng, J.; Peng, Y.; He, C.; Long, X.; Li, P.; Chan, A. S C Polym. Int. 2003, 52, 1182. (21) Hao, L.-Y.; Zhu, C.-L.; Jiang, W.-Q.; Chen, C.-N.-; Hu, Y.; Chen, Z.-Y. J. Mater. Chem. 2004, 14, 2929. (22) Montagne, F.; Mondain-Monval, O.; Pichot, C.; Elaı¨ssari, A. J. Polym. Sci.: Polym. Chem. Ed. 2006, 44, 2642.
Langmuir, Vol. 23, No. 22, 2007 10941
Figure 1. Schematic illustration of the synthesis of PPyCOOHcoated, PNVP-stabilized Fe2O3-PS latex particles.
cyclic voltammetry, and magnetic properties. They were then rendered reactive toward proteins by activation of the surface carboxyl groups using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The soformed activated particles were further incubated with biotin in order to monitor the formation of interfacial bonds between the carrier (N-succinimidyl ester functionalized polypyrrole particles) and the biomolecule. The assembly properties of these latex particles onto hydrophilic glass plates were then studied in the presence and absence of a magnetic field, as well as the electrochemical properties of PSmag-PPyCOOH particle-coated ITO electrodes. 2. Experimental Section 2.1. Materials. The super-paramagnetic latex particles (PSmag) were from Ademtech S. A. (Pessac, France). They are composed of magnetic core-containing iron oxide nanoparticles and polystyrene shell-bearing carboxylic functions (350 µmol.g-1). The zeta potential value of the latex suspension at pH 6, in 1 mM NaCl, was measured to be -35 mV, indicating that the magnetic latex particles are negatively charged. The PSmag latexes were washed before use and before all analyses. Poly(N-vinylpyrrolidone), with a nominal molecular weight of 360 000, was purchased from Aldrich and used without further purification. Pyrrole (Fluka) was purified by passing through a column of activated basic alumina (Acros) prior to use. The N-(2-carboxyethyl)pyrrole comonomer was synthesized as previously described in ref 9. N-(2-Cyanoethyl)pyrrole (Acros), FeCl3‚6H2O (Aldrich), biotin ethylenediamine (Sigma), and aminopropyltrimethoxysilane (Aldrich) were used without further purification. All aqueous solutions were prepared with deionized water. 2.2. Synthesis of Surface-Functionalized Polypyrrole-Coated Polystyrene Latex Particles. The coating procedure consists of the copolymerization of the monomers in the presence of PSmag latex particles. In order to improve the colloidal stability of the conducting polymer-coated PSmag latex, the PSmag latex particles were previously stabilized by dissolving 0.02 g of PNVP in 3 mL of the latex dispersion (0.12 g dry weight). After 4 h, 0.09 g of FeCl3‚6H2O and a mixture of 8.25 µL of pyrrole and 18 mg of pyrroleCOOH (50/50 molar ratio) were added to the vigorously stirred latex suspension and left to react at room temperature for 12 h. The resulting particles were then washed by five magnetic separation-redispersion cycles. The redispersion was performed in deionized water. It is worth noting that the magnetic properties of the particles facilitate their washing and concentration. The poly(pyrrole/pyrroleCOOH)-coated PSmag particles are abbreviated PSmag-PPyCOOH. 2.3. Activation and Reactivity of PSmag-PPyCOOH. Activation of the PSmag-PPyCOOH latex particles was achieved by dissolving 10-4 M, 10-3 M, and 10-2 M of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and, respectively, 2.10-4 M, 2.10-3 M, and 2.10-2 M of N-hydroxysuccinimide (NHS) in the aqueous PSmag latex suspension (12 mg.mL-1). After 2 h incubation, the activated latex particles were washed via magnetic separation-redispersion cycles. The activated ester-functionalized latex particles are abbreviated by PSmag-PPyNSE, where NSE stands for N-succinimidyl ester.
10942 Langmuir, Vol. 23, No. 22, 2007 Reactivity of the activated PSmag-PPyNSE latex particles was evaluated using biotin ethylenediamine. A solution of biotin ethylenediamine in PBS buffer pH 7.4 was added to the activated latex suspension (12 mg dry weight in 1 mL). The final biotin ethylenediamine concentrations were 5 × 10-4 M and 10-2 M. The reaction was carried out under gentle mixing during 16 h at room temperature. The product was cleaned via magnetic separationredispersion repetitive cycles in order to remove unreacted biotin ethylenediamine. The cleaned final product was then dried under vacuum. 2.4. PSmag-PPyCOOH Particle Assemblies. The glass plates were rendered hydrophilic following the procedure described by Yan et al.23 The colloidal assemblies were obtained by placing a 40 µL drop of the colloidal suspension of a given concentration on the hydrophilic glass plates and carefully spreading it to fully cover the substrate surface. The experiments performed in the presence of a magnetic field were performed by placing the magnetic latex suspension-covered glass plates above a permanent magnet. 2.5. Electrochemistry. For electrochemical analyses of the PSmagPPyCOOH latex particles in suspension, a carbon graphite disk (3 mm diameter) was used as a voltammetric working electrode. A graphite carbon paper and the SCE electrode were used as a counter electrode and a reference electrode, respectively. LiClO4 (0.1 M) was added to the latex suspension, as a supporting electrolyte. For the electrochemical analyses of PSmag-PPyCOOH assemblies, 40 µL of the latex suspension was dropped on an ITO electrode, placed above a permanent magnet. After allowing it to dry, the latex particlemodified ITO electrode served as the working electrode. 2.6. Analytical Techniques. Transmission electron microscopy (TEM) micrographs were obtained using a JEOL JEM 100CXII UHR operating at 100 kV. Solutions containing the magnetic latex particles were cast onto Formvar-coated copper grids, and the solvent was allowed to evaporate. Scanning electron micrographs (SEM) were obtained with a Cambridge 120 that is completely controlled from a computer workstation. The filament is a zirconated tungsten, and the accelerating voltage was set at 20 kV. All specimens were coated with gold prior to analysis in order to avoid or limit static charging effects. A Quantum Design MPMS-5S SQUID magnetometer was used for magnetic characterization. The thermal ZFC (zero-field cooling) and FC (field cooling) dc-susceptibility, χ, variations were measured under a magnetic field of 200 Oe in the temperature range 4.2-320 K. All the measurements were conducted on freshly produced particles slightly compacted in a plastic sampling tube in order to prevent their physical movement during the experiments. FT-IR spectra were recorded using a Nicolet Magna 550 series II instrument. Spectra were typically averaged over 20 scans at 4 cm-1 resolution. X-ray photoelectron spectroscopy (XPS) measurements were performed using a Thermo VG ESCALAB 250 instrument equipped with a monochromatic Al KR X-ray source (1486.6 eV). The X-ray spot size was 650 µm. The pass energies were set at 150 and 40 eV for the survey and the narrow scans, respectively. Charge compensation was achieved with a combination of electron and argon ion flood guns. The energy and emission current of the electrons were 4 eV and 0.35 mA, respectively. For the argon gun, the energy and the emission current were 0 eV and 0.1 mA, respectively. The partial pressure for the argon flood gun was 2 × 10-8 mBar. These standard conditions of charge compensation resulted in a negative but perfectly uniform static charge. Data acquisition and processing were achieved with the AVantage software, version 2.20. Spectral calibration was determined by setting the main C1s component at 285 eV. The surface composition was determined using the integrated peak areas and the corresponding Scofield sensitivity factors corrected for the analyzer transmission function.
3. Results and Discussion 3.1. Chemical Composition, Morphology, and Magnetic Properties of PSmag-PPyCOOH Particles. Photographs of (23) Yan, Q.; Zhou, Z.; Zhao, X. S. Langmuir 2005, 21, 3158.
Mangeney et al.
Figure 2. Digital photographs of (a) uncoated PSmag latex, (b) coreshell PSmag-PPyCOOH latex particles, and (c) the core-shell particles in the presence of a magnet.
Figure 3. SEM micrographs of (a) uncoated PSmag latex and (b) core-shell PSmag-PPyCOOH latex particles.
PSmag and PPyCOOH-coated PSmag latexes are displayed in Figure 2. The original light brown uncoated latex particles (Figure 2a) turned to deep black after the copolymerization process of pyrrole and pyrroleCOOH has occurred, therefore reflecting the covering of the magnetic polystyrene particles by the conducting polymer overlayer (Figure 2b). Moreover, the PSmag-PPyCOOH latex particles readily separate when the sample is placed near a permanent magnet, therefore demonstrating qualitatively that the new conducting polymer particles have magnetic properties (see Figure 2c). Figure 3 displays SEM micrographs of PSmag and PPyCOOHcoated PSmag latex particles. Particles are spherical and have polydisperse size distribution. The number average diameter (Dn) and the polydispersity parameter (Dv/Dn) of particles were measured directly from the SEM images. In the common procedure, the sizes of 500 particles were measured and the values were averaged. The Dn and Dv were calculated from the following equations:
Fe2O3-PS/PPy Core/Shell Particles
Langmuir, Vol. 23, No. 22, 2007 10943
Dn )
∑iNiDi ∑iNi
Dv )
∑iNiDi4 ∑iNiDi3
and
(1)
where Di means the diameters of individual particles and Ni refers to the number of particles corresponding to the diameters. The number average particle diameter increases from around 190 nm (Dv/Dn ) 1.1) for PSmag particles to around 220 nm (Dv/Dn ) 1.1) for PSmag-PPyCOOH particles. The difference between these two diameters (30 nm) gives an estimation of the conductive overlayer thickness, i.e., around 15 nm. According to Yamamoto and co-workers, PNVP chains with a molecular mass of 360 000 have an adsorbed layer thickness of ca. 20-30 nm.24 Therefore, the conducting polymer overlayer is smaller or comparable to the thickness of the adsorbed PNVP layer allowing the stabilization mechanism imparted by the PNVP chains to be efficient. It is also possible that the surface charges of the carboxyl groups account for electrostatic stabilization. TEM microscopy allowed to compare the particle morphology of PSmag and PSmag-PPyCOOH particles taken from the same batches as the particles imaged by SEM. As shown in Figure 4a, the latex particles are constituted of two distinct components: a black core of iron oxide nanoparticles (average diameter 130 nm) and a gray shell of polystyrene (average thickness 30 nm). The comparison between PSmag and PSmag-PPyCOOH particles evidences two major modifications of the particles morphology. First, some holes appear inside the magnetic core after the polymerization process. This can be explained by the strong acidity of the synthesis medium during polymerization that etches partly the iron oxide nanoparticles. Second, the particles surface morphology is modified with a noticeable roughening of the shell. These modifications in the surface morphology and roughness of the latex particles reflect the presence of a PPy shell around the PSmag particles. Such particle surface roughening is in line with previously published data25,26 on PPy-coated nonmagnetic PS particles. From the conducting polymer shell thickness (dTEM), TEM can be used as a semiquantitative method for the determination of the mass fraction of the PPyCOOH copolymer, as reported in ref 25. Indeed, assuming a uniform coating of PPyCOOH at the surface of PSmag particles, one can relate the thickness x of conducting copolymer overlayer to the main fraction of the PSmag core and the conducting shell using
x)r×
{[( ) ] } M2F1 +1 M1F2
1/3
-1
(2)
where M is the mass fraction, F the density,28 and r the radius of the core. The subscripts 1 and 2 refer to PSmag and PPyCOOH, respectively. One can rewrite eq 2 as
( ) {[(
F2 M2 ) × M1 F1
x+r3 -1 r
)]
}
(3)
(24) Liu, C. F.; Moon, D. K.; Maruyama, T.; Yamamoto, T. Polym. J. 1993, 25, 775. (25) Lascelles, S. F.; Armes, S. P. J. Mater. Chem. 1997, 7, 1339. (26) Benabderrahmane, S.; Bousalem, S.; Mangeney, C.; Azioune, A.; Vaulay, M.-J.; Chehimi, M. M. Polymer 2005, 46, 1339. (27) Long, Y.; Chen, Z.; Shen, J.; Zhang, Z.; Zhang, L.; Xiao, H.; Wan, M.; Duvail, J. L. J. Phys. Chem. B 2006, 110, 23228.
Figure 4. TEM micrographs of (a) uncoated PSmag latex particles and (b) core-shell PSmag-PPyCOOH latex particles.
Figure 5. XPS survey scans of (a) PSmag and (b) PSmag-PPyCOOH particles. The dashed area in (a) accounts for inelastically scattered Fe2p photoelectrons.
x is estimated from the difference in radii between the uncoated PSmag core and the PPyCOOH-coated particles. Combining the calculated M2/M1 ratio with (M2 + M1 ) 1), one can deduce the mass fraction of PPyCOOH, which is around 31%. This experimental mass fraction value compares well with the theoretical reactive polypyrrole loading value, i.e., 28%, thus indicating the absence of soluble fraction of conducting polymer. XPS spectroscopy proved to be useful to verify the presence of the PPyCOOH coating on the surface of the latex particles. Figure 5 displays the XPS survey spectra of PSmag and PSmagPPyCOOH particles. For PSmag, the main peaks are C1s and O1s centered at 285 and 532 eV, respectively. The Fe signal is absent from the survey spectrum (Figure 5a), indicating that the (28) The assumed density of PPyCOOH is based on the values of 1.53 and 1.45 g.cm-3 for polypyrrole and poly(pyrrole-3-acetic acid), respectively. Since we are dealing with a copolymer of pyrrole and PyCOOH, it follows that the density of the conducting polymer can be assumed to be ca. 1.49. For PSmag, the density is equal to 2 g.cm-3.
10944 Langmuir, Vol. 23, No. 22, 2007
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Table 1. Apparent Surface Chemical Composition as Determined by XPS samples
C
N
O
Fe
PSmag PSmag-PPyCOOH bulk PPyCOOHa
86 74 74
0 9 13
14 16 13
0 ∼1 -
a Theoretical values calculated for bulk PPyCOOH, using a brut formulas C11N2O2 corresponding to 50% pyrrole and 50% pyrroleCOOH.
underlying Fe2O3 is not probed by XPS. As XPS is a highly surface-specific technique with a typical analysis depth of ∼10 nm (clearly lower than the thickness of the polystyrene shell ca. 30 nm), it is reasonable that Fe2O3 is not detected. It thus provides experimental proof of the uniformity of the PS layer coating the magnetic core. However, it is worth noting that the survey scan in Figure 5a exhibits an increase in the background beyond the Fe2p peak position (lower kinetic energy side). In Figure 5a, the increase in the background is represented by a dashed area that holds for inelastically scattered Fe2p photoelectrons. This spectral region is actually at the low kinetic energy side of the parent Fe2p peak position and accounts for energy loss experienced by the emitted Fe2p electrons that escaped the ∼30 nm-thick PS shell. The absence of Fe2p together with the detected inelastically scattered Fe2p photoelectrons is a strong supporting evidence that iron oxide is buried well below the free surface of PS latex and confirms the structure of the particles as imaged by TEM (see Figure 4). In the PSmag-PPyCOOH spectrum, a new peak appears at 399.7 eV, assigned to photoelectrons originating from the N1s energy level. This signal, which is not observed in the spectrum of the underlying PSmag -latex particles, is a fingerprint of the PPyCOOH overlayer around the latex particles. Furthermore, new minor peaks appear in the PSmagPPyCOOH spectrum: Cl2p (at 198 eV) and Fe2p3/2 (at ∼710 eV), assigned respectively to the chloride dopants and to the insertion of iron in the form of FeCl2 resulting from the reduction of the oxidizing agent FeIII. Table 1 reports the surface atomic composition of PSmag and PSmag-PPyCOOH latex particles. The high oxygen content in uncoated PSmag particles arises from the surface carboxyl groups that ensure the electrostatic stability of the colloid. As previously observed, the covering of PSmag by the PPyCOOH layer induces the appearance of nitrogen in the surface chemical composition of the particles. This results in a depression of the carbon content by comparison to uncoated PSmag. It is interesting to note that the carbon content of PSmagPPyCOOH is identical to the theoretical value calculated for a bulk powder PPyCOOH with 50% pyrrole and 50% pyrroleCOOH. It follows that, on the basis of the carbon content, the underlying PSmag does not contribute to the surface composition. Indeed, the PPyCOOH thickness (ca. 15 nm), determined previously by TEM, is higher than the sampled layer (ca. 5-10 nm), hence the complete screening of PSmag by the uniform PPyCOOH coating. The contribution of PNVP steric stabilizer should not affect the composition in atomic percentages, because PPyCOOH copolymer has almost the same stoichiometric formula (C5.5NO) as PNVP (C6NO). In Table 1, one can note, however, that the experimental N and O contents slightly differ from the expected ones. This could be due to surface oxidation of the polypyrrole shell (see comments of Figure 7 below). Figure 6 shows the high-resolution N1s spectrum of PSmagPPyCOOH. It is similar to that observed for bulk polypyrrole, in agreement with the literature. It can be fitted with three components centered at ca. 398.9, 400.1, and 401.2 eV assigned to the imine NdC group, the NsH bond in the pyrrole repeat
Figure 6. Peak-fitted N1s core-line spectrum of PSmag-PPyCOOH particles.
unit, and the oxidation state of nitrogen CsN+ in doped polypyrrole, respectively. It is noteworthy that the N1s signal arising from PNVP should be found at 399.8 eV but could not be resolved as a separate component. The high-resolution C1s spectra for the uncoated and polypyrroleCOOH-coated PSmag particles are shown in Figure 7. For PSmag, the C1s region exhibits two main components centered at 285 and 286.6 eV and a minor one centered at 289 eV. The two low binding energy (BE) components can be assigned to C-C/C-H and oxidized carbon atom functionalities, respectively, while the minor component at 289 eV is due to the polystyrene surface carboxyl groups. After coating by polypyrrole overlayers, the PSmag-PPyCOOH particles exhibit a C1s narrow region that is very distinct from that of the PSmag latex and which can be fitted with four components. These are centered at 285 (pyrrole β carbon and underlying polystyrene), 286.4 (pyrrole R carbon and surface oxidation of carbon atoms from the shell), 288 (PNVP NsCd O), and 289 eV (carboxyl groups). Table 2 summarizes the results of the C1s peak fitting. It is noteworthy that the carboxyl component intensity has been multiplied by more than 5 compared to the underlying PSmag latex. The drastic change of the C1s peak from PSmag to PSmag-PPyCOOH latex provides strong evidence that the surface of this material is essentially PPyCOOH. The plots of the FC and ZFC magnetic susceptibilities, χ, versus temperature of the PSmag and PSmag-PPyCOOH particles are given in Figure 8. The obtained curves are typical of a superparamagnetic behavior for the two samples. Indeed, while the ZFC-χ(T) curves show a maximum at T ) 71 K for PSmag and at T ) 61 K for PSmag-PPyCOOH, the FC-χ(T) curves show a plateau bellow these temperatures. The temperatures of these maxima of ZFCχ(T) correspond to a critical temperature called blocking temperature TB, below which the ferrimagnetic order of the magnetic cores (Fe2O3) can be observed. The weak variation of the TB value (from 71 to 63 K) shows that the recovering of the magnetic latex particles by the conducting polymer shell does not affect significantly the intrinsic magnetic properties of the core. It appears clearly that the magnetic properties of the synthesized composites are mainly driven by the superparamagnetic character of the iron oxide nanoparticles. Moreover, using the experimental FC-χ(T) value (χ being expressed by gram) at a given temperature T < TB, for instance, T ) 5 K, it is possible to estimate x the amount (weight percent) of the
Fe2O3-PS/PPy Core/Shell Particles
Langmuir, Vol. 23, No. 22, 2007 10945
Figure 7. Peak-fitted C1s core-line spectra of PSmag and PSmag-PPyCOOH particles.
Figure 8. Magnetic susceptibilities of (a) PSmag and (b) PSmagPPyCOOH particles as a function of temperature in the FC and ZFC modes. Table 2. Peak Fitting of C1s Spectra for PSmag and PSmag-PPyCOOH Latex: Binding Energies (eV) of the Various C1s Components and Their Corresponding Relative Intensities (%) between Brackets CsC, CdC PSmag PSmag-PPyCOOH
285.0 (73) 285.0 (58)
-CsO
NsCdO
OsCdO
286.6 (25) - (0) 288.9 (2) 286.4 (23) 287.9 (8) 289.0 (11.0)
PPyCOOH shell, stabilized by PNVP, in the PSmag-PPyCOOH particles
χPSmag-PPyCOOH ) (1 - x - )χPSmag + xχPPyCOOH + χFe2+ (4) where χPSmag, χPPyCOOH, and χFe2+ are the total magnetic susceptibilities of the PSmag core particles (superparamagnetic contribution in majority), the PNVP-stabilized PPyCOOH shells and the isolated doping iron cations, respectively, and the weight percent of the initially introduced iron salt in the polypyrrolecoating process (less than 1 wt %). χPSmag is the measured value on the PSmag particles. Neglecting the diamagnetic contribution of PPyCOOH and PNVP, χPPyCOOH is the sum of a Pauli-like (independent of T) and a Curie-like (proportional to 1/T) paramagnetic susceptibilities which are originated from the delocalized π-radicals in the π-conjugated systems, i.e., polarons. These magnetic signals are usually very weak27 compared to that of the superparamagnetic iron oxide particles, and can be neglected in a first approximation, in the x value estimation. By also neglecting the Curie-like paramagnetic signal of the Fe2+ free
Figure 9. Cyclic voltammogram of the PSmag-PPyCOOH latex dispersed in 0.1 M LiClO4 (potential scan rate V ) 100 mV.s-1). Inset shows the dependence of the anodic peak current, Ia, on scan time, V.
cation, since they are present in a very low amount in the final composite, x is found to be about 31 wt %. This experimental mass fraction value compares well with the value extracted from TEM images (see above) as well as with the theoretical PNVPstabilized PPyCOOH loading value (28%). The colloidal stability of PNVP-stabilized PSmag-PPyCOOH latexes was assessed qualitatively. The PPyCOOH coating slightly affects the colloidal stability of the functionalized PSmag latex seed particles. Given the relatively high Hamaker constant reported for polypyrrole,29 it is perhaps not surprising that the polypyrrole overlayer can interfere with the steric stabilization mechanism responsible for maintaining the colloidal stability of the latex particles. However, as the polypyrrole overlayer is sufficiently thin (ca. 15 nm) relative to the stabilizer layer thickness (ca. 20-30 nm24), it is possible to minimize destabilization of the coated latex particles. 3.2. Electrochemical Properties of PSmag-PPyCOOH Particles. Figure 9 shows the cyclic voltammogram of the PSmagPPyCOOH latexes, in LiClO4 (0.1 M) containing water suspension. The latex dispersions were swept through a potential of -0.5 to +0.3 V versus SCE at various scan rates. The CV data indicate that the PPyCOOH-coated PSmag particles are electro(29) (a) Markham, G.; Obey, T. M.; Vincent, B. Colloids Surf. 1990, 51, 239. (b) Chehimi, M. M.; Lascelles, S. F.; Armes, S. P. Chromatographia 1995, 41, 671.
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Mangeney et al. Table 3. Apparent Surface Chemical Composition of PSmag-PPyCOOH Particles after Activation by EDC/NHS, as Determined by XPS samples -4
[EDC/NHS] 10 M [EDC/NHS] 10-3 M [EDC/NHS] 10-2 M
C
N
O
(N + O)/C
74 71 69
8 8 9
18 21 22
0.35 0.41 0.45
Table 4. Apparent Surface Chemical Composition of Activated PSmag-PPyNSE Particles before and after Incubation with Biotin, as Determined by XPS samples
C
N
O
Fe
S
PSmag-PPyNSEa Biotin-PSmagPPyNSE Pure Biotinb
69 66 63
8 9 21
21 24 11
2 ∼0 -
0 1 5
a PS -3 M of mag-PPyNSE were obtained from reaction with 10 NHS/EDC. b Theoretical atomic percents calculated from biotin stoechiometry C12N4O2S.
Figure 10. FTIR spectra in the 1400-2000 cm-1 region of (a) PSmag-PPyCOOH latex particles, (b) EDC/NHS activated PSmagPPyNSE particles, and (c) biotinylated PSmag-PPyNSE latex particles. Inset shows, schematically, the activation of the particle surface carboxyl groups, using EDC/NHS, and reactivity of the resulting activated ester groups toward amine-bearing molecules.
active in aqueous medium. However, the electroactivity is rather low, and the anodic and cathodic waves are ill-defined. In the inset of Figure 9, the anodic peak currents were plotted against the scan rate, V: it is observed to be proportional to V, suggesting a surface wave by adsorption. Such a behavior is in agreement with previously published results30 on the redox reactions of polyaniline-coated latex suspensions where digital photographs confirmed the adsorption of the latex particles on the electrode. 3.3 Activation and Biotinylation of PSmag-PPyCOOH Particle. The carboxyl surface groups of PSmag-PPyCOOH particles have to be activated in order to be reactive toward biomolecules. The activation step, described in the inset of Figure 10, consisted of reacting the PSmag-PPyCOOH suspension with N-hydroxysuccinimide (NHS), in the presence of 1-ethyl-3-(3dimethylaminopropyl)carbodiimide (EDC) for 2 h. Then, the activated particles were purified by five cycles of magnetic separation-redispersion. The dried products were characterized by FTIR and XPS. IR spectra, in the 1400-2000 cm-1 region, of PSmag-PPyCOOH particles before and after activation by EDC/NHS are shown in Figure 10. The spectrum of the PSmagPPyCOOH particles displays two intense bands: one at 1560 cm-1, characteristic of polypyrrole, and one at 1650 cm-1 assigned to the carboxyl groups. The steric stabilizer is also detected (at 1605 cm-1), showing that it remains adsorbed on the PS surface even after coating by the conducting polymer layer. This observation is in agreement with the XPS data (see Table 2) and with previously reported microanalysis data on polypyrrole-coated polystyrene latex particles,25 which allowed estimation of an adsorbed amount of PNVP stabilizer of ca. 4.5 mg.m-2. After incubation with increasing amounts of EDC/NHS, clear modifications of the PSmag-PPyCOOH spectrum are observed: three new bands appear. The band at ca. 1650 cm-1 may be attributed to the OsH bending of physically adsorbed water, while the intense band at 1720 cm-1 can be assigned to the succinimidyl ester CdO stretching vibration and the weak one at 1820 cm-1 is due to the pyrrolidone CdO stretching vibration. Furthermore, the intensities of these last two bands increase progressively with the concentration of NHS and EDC, indicating (30) Aoki, K.; Chen, J.; Ke, Q.; Armes, S. P.; Randall, D. P. Langmuir 2003, 19, 5511.
the transformation of the surface carboxyl groups into succinimidyl ester groups (NSE). Beyond a concentration of about 10-2 M in EDC/NHS, the intensities of the succinimidyl ester bands no longer change, suggesting the complete transformation of the active sites present at the surface of the latex particles. The resulting N-succinimidyl ester-activated particles are hereafter abbreviated by PSmag-PPyNSE. XPS was used to characterize the surface composition of the activated particles. Table 3 shows that, after esterification of the magnetic, conductive particles, the (N + O)/C atomic ratio increases as the initial quantity of EDC/NHS increases in the reaction medium. The overall elemental signal evolution (with a decrease in carbon content and an increase in nitrogen) can be explained by the transformation of pyrroleCOOH (with a theoretical (N + O)/C atomic ratio of 0.43) into succinimidyl ester pyrrole (with a theoretical (N + O)/C atomic ratio of 0.54). Although adsorbed water was detected by IR, under XPS high vacuum conditions it is unlikely that water would remain sorbed at extents above the detection limit of the apparatus. The high-resolution C1s spectrum (not shown) changes also after reaction with NHS/EDC with a broadening of the signal at high binding energy due to the introduction of CdO pyrrolidone groups borne by the succinimidyl ester functions. These activated PSmag-PPyNSE particles were then evaluated for reaction with biotin, chosen as a model biomolecule with a strong affinity toward the protein avidin (KD ≈ 10-15). PSmag-PPyNSE particles were incubated with biotin for 12 h and then purified by five cycles of magnetic separationredispersion. The IR spectrum of the dried products is shown in Figure 10c. The ester band at 1720 cm-1 has almost disappeared after incubation with biotin, indicating the release of the succinimidyl ester groups. Simultaneously, a new band appears at 1630 cm-1, which can be assigned to amide groups resulting from the interfacial link between biotin residues and the reactive latex surface groups. The XP spectra of the biotin-incubated PSmag-PPyNSE particles also displays modifications compared to the unreacted particles. The atomic surface compositions in Table 4 indicate that PSmag-PPyNSE particles were indeed biotinylated, as they exhibit uptake of sulfur and a slight decrease of the carbon content. Actually, if one considers 12 carbons for 1 sulfur in biotin, it is possible to estimate a biotin content of ca. 20% in the surface layer of the latex particles. Such a high value suggests that the modification occurs not only on the extreme surface of the particles, but also within the conductive polymer layer. It is likely that the large amount of carboxyl groups in this layer
Fe2O3-PS/PPy Core/Shell Particles
Langmuir, Vol. 23, No. 22, 2007 10947
Figure 12. SEM micrographs at various magnifications of PSmagPPyCOOH assemblies on hydrophilic glass plates. No magnetic field is applied.
Figure 11. SEM micrographs at various magnifications of PSmag assemblies on hydrophilic glass plates. No magnetic field is applied.
makes it swell somewhat in water, thus allowing the diffusion of biotin inside.
4. Assemblies of PSmag-PPyCOOH Particles onto Hydrophilic Glass We further explored the electrostatic assemblies of PSmagPPyCOOH particles on glass plates, in the presence and absence of a magnetic field. For the experiment undertaken in the absence of a magnetic field, hydrophilic glass plates were placed horizontally, and 40 µL of the PSmag-PPyCOOH colloidal suspension was dropped on the substrate and carefully spread to fully cover the substrate. The suspension-covered plates were then left to dry at ambient conditions (temperature 20 °C). After about 1.5 h, a thin colloidal crystal film was formed on the substrate. During the growth of the colloidal crystal film, an air flow was applied on the drop. Without applying such an air flow, a void was observed in the center of the film or the substrate. By using a gas flow, the void can be removed or at least positioned
at the edge of the substrate. The observation of a void area forming on the substrate during PS colloid crystallization has already been observed by Zhao et al.23 It was interpreted by a mechanism of inward-growing self-assembly from the periphery toward the center of the spread suspensions on the horizontal substrate. Figure 11 shows the SEM images of colloidal films deposited on glass substrates from PSmag colloidal suspensions of 0.8 wt % concentration, viewed at different magnifications. When viewed at a low magnification (Figure 11a,b), it is seen that the films are composed of many domains separated by cracks. The domain size can be as large as 20 × 30 µm2. The formation of the cracks is most likely due to the shrinkage of the self-assembled spherical particles during the drying process. When viewed at a larger magnification (Figure 11c), the uncoated PSmag spheres form a fairly organized 3D network with the particles so closely packed that one observes that they are no more spherical but slightly distorted. In contrast, the colloidal crystal of PSmag-PPyCOOH spheres (see Figure 12) shows particles which are relatively distant from each other and which are organized as fairly ordered close-packed structures. A certain degree of disorder, probably due to particle polydispersity, site randomness, and dislocation, is also observed. The sticking behavior of the PSmag particles arises from the strong magnetodipole interactions between them, while for PSmag-PPyCOOH particles, the conducting polymer shell as well as the PNVP stabilizer probably provide a steric layer that prevents the particles from gluing together. The presence of a magnetic field, created by a magnet placed under the hydrophilic glass plates, has a dramatic effect on these assemblies. As can be seen on the SEM micrographs displayed in Figure 13, the magnetic field induces new organizations of the spheres: at low magnification, fernlike structures are observed. When viewed at a high magnification (Figure 14), it is seen that these fernlike structures are made of particle chains gathered in hedgehog-like architectures. The magnetic field generates a magnetic moment in each particle, and depending on the direction of the field, the dipolar interaction leads to aggregation of the particles into a bunch of regular one-particle-thick chains. As previously observed on cobalt nanoparticle assemblies,31 it is expected that the formation of fernlike structures arises from a progressive collapse of part of the columns because of the waves induced by capillary forces during the evaporation process. (31) Germain, V.; Pileni, M. P. J. Phys. Chem. B 2005, 109, 5548.
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Mangeney et al.
Figure 13. SEM photographs of the assemblies of PSmag-PPyCOOH on hydrophilic glass plates, under a magnetic field created by a permanent magnet.
Figure 14. SEM photographs of the assemblies of PSmag-PPyCOOH on hydrophilic glass plates, under a magnetic field created by a permanent magnet.
Figure 15. Cyclic voltammogram of the PSmag-PPyCOOH particlecoated ITO electrode in aqueous solution containing 0.1 M LiClO4 (potential scan rate V ) 100 mV.s-1). Inset shows the dependence of the anodic peak current, Ia, on scan time, V.
By taking a square area (S ) 1 µm2) inside a fernlike structure and calculating the number of latex particle columns (Ncol) inside this area as well as the average number of particles inside a column (Npart), it is possible to get an estimation of the increase in specific area obtained in such structures
Ncol × [Npart × (4πR2)]/S where R is the particle diameter. This calculation gives an approximate 10-fold increase of the specific area on the fernlike structures. Such high specific area makes these modified substrates promising candidates for high-sensitivity biosensing devices.
Nevertheless, it is not clear whether the magnetic latex filaments are irreversibly stuck or if the drying process glues weakly the latex particles together. Work is in progress to probe the bending rigidity of the filaments. The electrochemical activity of the PSmag-PPyCOOH assemblies deposited on an ITO electrode was studied in 0.1 M aqueous solution of LiClO4. The assemblies exhibit good electrochemical stability, and the cyclic voltammograms of the PSmag-PPyCOOH-coated ITO electrodes (see Figure 15) have a couple of broad oxidation and reduction waves at -0.12 and -0.20 V versus SCE, respectively. Such values are in agreement with previously published results on derivatized pyrrole copolymers.32 As reported in the inset of Figure 15, the wave currents have linear relationships with potential scan rates in the range 20-200 mV.s-1, indicating that the mass and electron transfers are fixed on the electrodes. Furthermore, the particle films can be cycled repeatedly between the conducting (oxidized) and insulating (neutral) state without significant decomposition.
5. Conclusion New magnetic Fe2O3-polystyrene-PPy latex particles (PSwere prepared and characterized in terms of chemical composition, reactivity, morphology, and magnetic properties. TEM showed an increase of the latex particle diameter from 190 to 220 nm when covered by the conducting polymer layer, thus indicating a PPyCOOH shell thickness of ∼15 nm. The presence of the conducting polymer shell does not affect the magnetic properties of the latex particles, PSmag and PSmagPPyCOOH both showing superparamagnetic behavior. Electrochemical data obtained from the LiClO4-containing PSmagmag-PPyCOOH)
(32) Korri-Youssoufi, H.; Makrouf, B.; Yassar, A. Mater. Sci. Eng. C 2001, 15, 265.
Fe2O3-PS/PPy Core/Shell Particles
PPyCOOH suspensions confirmed that the latex particles are redox-active, with peak currents which are adsorption-limited. The carboxyl groups present at the PSmag-PPyCOOH particle surface were transformed into activated esters using EDC/NHS. The resulting particles, PSmag-PPyNSE, were evaluated as bioadsorbents and incubated with biotin, chosen as a model biomolecule. XPS and IR spectroscopy showed that biotin was covalently immobilized at the PSmag-PPyCOOH particle surface, through the formation of interfacial amide bonds. These PSmagPPyCOOH latexes could thus be used, after activation with EDC/ NHS, for the covalent attachment of biomolecules, making these supports suitable for biomedical applications. The self-assembling property of PSmag-PPyCOOH particles onto the surface of hydrophilic glass plates is dependent on the
Langmuir, Vol. 23, No. 22, 2007 10949
presence or absence of a magnetic field. When no magnetic field was applied, colloidal crystals of latex particles were obtained. In contrast, the magnetic field created by a magnet, located under the glass plates, induced original architectures such as regular one-particle-thick chains gathered in hedgehog-like structures. Furthermore, the PSmag-PPyCOOH particles, assembled under a magnetic field, on ITO electrodes were shown to be electroactive with a high stability under potential cycling. From the above, it can be concluded that the bioreactivity of the assembled particles together with the 10-fold increase in the specific area make these new magnetic PSmag-PPy particles promising materials for the design of new biosensing devices. LA700492S
