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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, Universite´ Paris 7 - Denis Diderot, CNRS (UMR 7086), 1 rue Guy de la Brosse, 75005 Paris, France, Laboratoire de Chimie des Mate´ riaux, Faculte´ 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 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 sterically stabilized polypyrrole latex particles by the introduction 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 development 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, Universite ´ Paris 7 - Denis Diderot, CNRS (UMR 7086). ‡ Laboratoire de Chimie des Mate ´ riaux, Faculte´ 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.
protein immobilization. However, the formation of a Schiff base is reversible (at pH ≈ 9) and thus requires stabilization by, for example, sodium cyanoborohydride.3 In the early 1990s, Armes and co-workers synthesized a second class of colloidal polypyrrole particles by polymerizing pyrrole in the presence of ultrafine inorganic particles such as silica.4 It was shown that these polypyrrole-silica nanocomposites had a “raspberry” morphology, with the silica particles being “glued” together by the precipitating polypyrrole.5-7 Armes et al.5,6 successfully synthesized carboxylic acid functionalized polypyrrole-silica nanocomposites by copolymerizing a functional pyrrolic comonomer (either 1-(2-carboxyethyl)pyrrole or pyrrole-3-acetic acid, respectively) with pyrrole during the nanocomposite synthesis. Amine-functionalized polypyrrole-silica nanocomposites were synthesized via two routes: (i) copolymerization of an N-substituted aminofunctional comonomer with pyrrole and (ii) the treatment of nonfunctionalized nanocomposites with 3-aminopropyltriethoxysilane (APTES). Since the latter nanocomposites 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 nanocomposites (PPy-silica, PPy-silica-COOH, and PPy-silicaNH2, 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 PPysilica-COOH and especially PPy-silica-NH2 particles.9 Pope et al.11 used N-carboxylic acid functionalized polypyrrole-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 chromatography.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 selfassembled monolayers (SAMs) on gold substrates after esterification by NHS in the presence of EDC. It was found that immobilization resulted in increased protein adsorption. 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 nanoparticles 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 spectroscopy (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 synthesized 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) Gu¨bitz, G. In Selective Sample Handling and Detection in HighPerformance 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-carboxyethylpyrrole) 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 Bu¨chner 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 Bru ¨ cker AC200 spectrometer 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 polypyrrolesilica-NHS nanoparticles in 0.1 M PBS at pH 7.4 according to the protocol previously described by Azioune et al.18 Briefly, 400500 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 “oneshot” 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.; Slomkowski, S. Langmuir 2002, 18, 1150.
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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 (Perkin-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.
resolution spectra. Under these conditions, the full width at halfmaximum (fwhm) of the C1s peak of the polypyrrole-silicaNHS 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 Nanocomposite. Figure 1 depicts the synthesis of the PyNHS 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 pyrrole3-acetic acid with pyrrole. This feed ratio gave nanoparticles that possessed a narrow size distribution and a reasonable degree of surface carboxylation. More importantly, 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 nanocomposite 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, whereas 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.
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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 conducted 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, respectively. 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 approximately 2, which is in good agreement with the chemical structure of this monomer (see Figure 1). The pyrroleNHS O1s peak (Figure 3c) was peak-fitted with two components centered at 532 and 534 eV. These components can be assigned to the carbonyl groups and the C-O-N oxygen atom from the pendent succinimide group. The CdO/C-O-N relative intensity is 3, as expected for the monomer structure. 2. Synthesis and Properties of the PolypyrroleSilica-NHS Nanocomposite. The novel N-ester-functionalized polypyrrole-silica nanocomposite particles have the same intense black color as the unfunctionalized homopolypyrrole-silica nanocomposites. This is characteristic of the highly conjugated structure of polypyrrole and, in the context of visual agglutination assays, makes these intrinsically chromogenic particles an interesting alternative to conventional extrinsically dyed polystyrene latex. The polypyrrole-silica-NHS nanoparticles have good colloidal stability, as judged from DCP measurements. The weight-average particle diameter (Dw) obtained by DCP was 210 ( 36 nm. Polydispersities are given by the ratio of Dw to the number-average diameter (Dn) and clearly show that these colloidal particles have relatively narrow size distributions. DCP results were found to be (21) Ba¨uerle, P.; Hiller, M.; Scheib, S.; Sokolowski, M.; Umbach, E. Adv. Mater. 1996, 3, 8.
Figure 3. XPS spectra of the N-succinimidyl ester pyrrole: (a) survey scan and (b) C1s and (c) O1s regions. Table 1. Peak Fitting Parameters for the C1s Core Level from the N-Succinimidyl Ester Pyrrole and N-Succinimidyl Ester Functionalized Polypyrrole-Silica Nanocomposite C-C/C-Hx (284.6 eV) C-N (286 eV) N-C ) O (288.1 eV) N-O-(C)O)-C (289.3 eV)
Py-NHS
PPy-NHS-silica
59.8 21.8 12.2 6.2
59.2 24.4 11.1 5.3
in fair agreement with the scanning electron microscopy (SEM) pictures (see Figure 4), indicating an average size of 175 ( 25 nm (average values calculated for 10 particles). Density measurements by helium pycnometry gave a value of 1.697 g cm-3 for the functionalized nanocomposites. Assuming the densities of 2.16 and 1.5 for silica and N-succinimidyl polypyrrole (PPy-NHS), it follows that the weight fraction of silica is ca. 30%. [The assumed density of PPy-NHS is based on the values of 1.53 and 1.45 for polypyrrole and poly(pyrrole-3-acetic acid),6 respectively. Since the pendent NHS group is much larger than the COOH, it is in principle expected that pure PPy-
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Figure 4. Scanning electron micrographs of the polypyrrolesilica-NHS particles.
Figure 6. XPS spectra of the polypyrrole-silica-NHS nanocomposite: (a) survey scan and (b) C1s high-resolution spectrum. Table 2. Surface Atomic Compositions of Polypyrrole-Silica Nanocomposites after the Immobilization of HSA in 0.10 M PBS at pH 7.4
Figure 5. FTIR spectrum of the polypyrrole-silica-NHS nanocomposite after 4 months of storage as an aqueous suspension at pH ∼ 2-3.
NHS (100% Py-NHS repeat units) would have a lower density than 1.45. However, since we are dealing with a copolymer of pyrrole and Py-NHS, it follows that the 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 17061709 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
materialsa
C
O
PPy-silica-NHS PPy-silica-NHS-HSA-10 µg/mL PPy-silica-NHS-HSA-20 µg/mL PPy-silica-NHS-HSA-50 µg/mL PPy-silica-NHS-HSA-150 µg/mL PPy-silica-NHS-HSA-300 µg/mL PPy-silica-NHS-HSA-500 µg/mL
37.8 50 56.8 64.2 58.3 69 60
42.5 39.5 32.3 24.3 27.5 20.3 25.6
N
Si
Na
5.8 13.8 0 4.2 5.4 0.7 5.4 4.6 0.9 6.3 4 1.2 7.7 4.6 1.5 6.4 3.7 1.3 7.1 4.4 3
a x µg/mL stands for the initial concentration of HSA used for 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 highresolution spectra obtained for the nanocomposite particles. 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 nanocomposite particles, like the unfunctionalized homopolypyrrole-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.
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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 components; 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 proportion by 11 (the number of carbon atoms in the PyNHS repeat units) and subtracting from the total C1s area, one obtains the contribution of pyrrole and pyrroleNHS repeat units to the total C1s peak area from the copolymer in the nanocomposite.] Thus, quantitatively, the nanocomposites have a silica-rich surface, on one hand, and the relative concentrations of pyrrole and Py-NHS at the particle surface correspond approximately to the initial comonomer feed prior to copolymerization, on the other hand. 3. Immobilization of Human Serum Albumin onto Polypyrrole-Silica-NHS Nanocomposites. The polypyrrole-silica-NHS nanocomposite was evaluated as a bioadsorbent of a model protein, HSA. The chemical reaction leading to protein immobilization on the nanocomposite is depicted in Figure 7. The NHS group reacts with the protein, and an amide bond is formed. The maximum amount of immobilized HSA determined using the Bradford method was approximately 300 mg g-1 for an initial protein concentration of 500 µg mL-1. This is a much higher loading than those found in our previous studies with either unfunctionalized polypyrrole-silica nanocomposite10 or powders.18 The N-succinimidyl ester functionalization of the polypyrrole-silica nanocomposites is thus a prequisite for an effective and massive attachment of proteins. In addition to the presence of surface reactive groups, the high specific surface area of the nanocomposite also plays an important role, and that is the reason adsorption is very high in milligrams of protein per gram of colloid. As outlined below, the adsorption in mg/m2 units remains, however, in line with the range of adsorption of proteins onto colloidal particles. Indeed, the nanocomposites have a raspberry shape that should result in a high specific surface area as for the unfunctionalized PPy-silica nanoparticles.4 Using the following equation established
Figure 8. XPS survey spectra of the polypyrrole-silica-NHS nanocomposite after incubation with human serum albumin at an initial concentration of (a) 50 µg/mL and (b) 500 µg/mL.
for nonporous particles, one can estimate the surface area (A):
A ) 3/(Fr) where F is the density (1.735 g cm-3) and r is the radius (75-100 nm) of the nanocomposites. The calculated value is ca. 20 ( 3 m2/g. However, Maeda and Armes4 have shown that a correction factor of at least 2.0 must be introduced in order to take into account the microporosity and roughness of the particles. We therefore estimate the specific surface area to be 40-50 m2/g. This value yields an immobilization of protein estimated to be 6-7.5 mg/ m2, which is in agreement with published data on monolayer adsorption of HSA with end-on conformation.23 The covalent attachment of the protein onto active esterfunctionalized polypyrrole-silica nanoparticles is due to the chemical reaction between amino groups of the protein and the pendent ester groups from the functionalized pyrrole units (see Figure 7). This is very likely and supported by the work of Korri-Youssoufi et al.24 These authors reported FTIR results on the immobilization of the oligonucleotide (ODN) onto 3-functionalized polypyrrole with ester groups that are in line with a covalent bond formation between the conducting substrate and the biological macromolecule. Indeed, during ODN attachment, they observed a disappearance of the frequencies associated with pyrrolidinedione at 1820 and 1786 cm-1 and the appearance of the frequency corresponding to the (23) Douglass, R. J.; Sasha, O.; Sharon, G. R. Langmuir 2000, 16, 5449. (24) Kourri-Youssoufi, H.; Makrouf, B. Anal. Chim. Acta 2002, 85, 469.
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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-immobilized 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
Figure 9. Plot of N/Si atomic ratio for the polypyrrole-silicaNHS 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 nanocomposite (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 nanocomposites 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.
Novel polypyrrole-silica nanocomposite particles bearing reactive surface N-hydroxysuccinimide functional groups were prepared in aqueous solution by copolymerization of pyrrole and N-esterified pyrrole (pyrroleNHS) 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 surfacefunctionalized polypyrrole-silica nanocomposites, together 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. Trippe´ 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 University Paris 7 - Denis Diderot for financial support through an invited professorship. LA030407S
