Probing the Conformation of Polyelectrolytes in Mesoporous Silica

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Langmuir 2008, 24, 4224-4230

Probing the Conformation of Polyelectrolytes in Mesoporous Silica Spheres Alexandra S. Angelatos, Yajun Wang, and Frank Caruso* Centre for Nanoscience and Nanotechnology, Department of Chemical and Biomolecular Engineering, The UniVersity of Melbourne, Victoria 3010, Australia ReceiVed NoVember 22, 2007. In Final Form: January 16, 2008 We report a fluorescence-based approach to probing the conformation of a macromolecule, poly(allylamine hydrochloride) (PAH), in bimodal mesoporous silica (BMS) particles. The method involves monitoring the fluorescent properties of the probe, 1,3,6,8-pyrenetetrasulfonic acid tetrasodium salt (4-PSA), upon electrostatic binding to PAH molecules adsorbed in the nanopores of the BMS particles. PAH infiltration into the BMS particles, quantified by thermogravimetric analysis and visualized by confocal laser scanning microscopy, was examined as a function of PAH adsorption time, PAH molecular weight, and the sodium chloride (NaCl) concentration and pH of the PAH adsorption solution. The conformation of PAH molecules in the nanopores was investigated by incubating the PAH-loaded BMS particles in 4-PSA and using the ratio of the excimer to monomer emission intensity to discern differences in the PAH conformation in the nanopores. Control experiments involving nonporous silica (NS) particles were also conducted to determine the extent to which the nanopores within the BMS particles influence the degree of PAH adsorption and the conformation of the adsorbed PAH molecules. The data indicate that PAH molecules adsorbed in the nanopores adopt a more coiled conformation than PAH molecules adsorbed on NS particles over a wide range of conditions. Further, the conformation of PAH molecules in the nanopores can be tuned by adjusting the NaCl concentration and/or pH of the PAH adsorption solution. 4-PSA titration experiments revealed that at saturation binding there are ca. 3.8 PAH monomer units per 4-PSA molecule. This study provides insights into macromolecule infiltration and conformation in nanopores, which are important for the application of mesoporous materials in the fields of adsorption/immobilization, catalysis, delivery, sensing, separations, and synthesis.

Introduction Mesoporous silicas (MSs), materials with pore diameters between 2 and 50 nm,1,2 are used extensively in adsorption/ immobilization,3-5 catalysis,3-7- delivery,8-12 sensing,13,14 and separations.15-18 They have also been widely used as templates to fabricate a variety of materials, including metal,19 metal oxide,20 carbon,21 and polymer22,23 replicas. MSs are attractive for such applications because they exhibit high surface areas, large pore * Corresponding author. E-mail: [email protected]. (1) IUPAC Manual of Symbols and Terminology. Pure Appl. Chem. 1972, 31, 578. (2) Kresge, C. T.; Leonowicz, M. E.; Roth, W. J.; Vartuli, J. C.; Beck, J. S. Nature 1992, 359, 710. (3) Hartmann, M. Chem. Mater. 2005, 17, 4577. (4) Yiu, H. H. P.; Wright, P. A. J. Mater. Chem. 2005, 15, 3690. (5) Wang, Y.; Caruso, F. Chem. Mater. 2005, 17, 953. (6) Maschmeyer, T.; Rey, F.; Sanker, G.; Thomas, J. M. Nature 1995, 378, 159. (7) Corma, A. Chem. ReV. 1997, 97, 2373. (8) Ahola, M.; Kortesuo, P.; Kangasniemi, I.; Kiesvaara, J.; Antti Yli-Urpo, A. Int. J. Pharm. 2000, 195, 219. (9) Arcos, D.; Ragel, C. V.; Vallet-Regı´, M. Biomaterials 2001, 22, 701. (10) Czuryszkiewicz, T.; Ahvenlammi, J.; Kortesuo, P.; Ahola, M.; Kleitz, F.; Jokinen, M.; Linden, M.; Rosenholm, J. B. J. Non-Cryst. Solids 2002, 306, 1. (11) Vallet-Regı´, M.; Ra´mila, A.; del Real, R. P.; Pe´rez-Pariente, J. Chem. Mater. 2001, 13, 308. (12) Radu, D. R.; Lai, C. Y.; Jeftinija, K.; Rowe, E. W.; Jeftinija, S.; Lin, V. S. Y. J. Am. Chem. Soc. 2004, 126, 13216. (13) Johnston, A. P. R.; Caruso, F. J. Am. Chem. Soc. 2005, 127, 10014. (14) Angelatos, A. S.; Johnston, A. P. R.; Wang, Y.; Caruso, F. Langmuir 2007, 23, 4554. (15) Han, Y. J.; Stucky, G. D.; Butler, A. J. Am. Chem. Soc. 1999, 121, 9897. (16) Stein, A.; Melde, B. J.; Schroden, R. C. AdV. Mater. 2000, 12, 1403. (17) Feng, X.; Fryxell, G. E.; Wang, L.-Q.; Kim, A. Y.; Liu, J.; Kemner, K. M. Science 1997, 276, 923. (18) Mercier, L.; Pinnavaia, T. J. AdV. Mater. 1997, 9, 500. (19) Liu, Z.; Sakamoto, Y.; Ohsuna, T.; Hiraga, K.; Terasaki, O.; Ko, C. H.; Shin, H. J.; Ryoo, R. Angew. Chem., Int. Ed. 2000, 39, 3107. (20) Dong, A.; Ren, N.; Tang, Y.; Wang, Y.; Zhang, Y.; Hua, W.; Gao, Z. J. Am. Chem. Soc. 2003, 125, 4976. (21) Ryoo, R.; Joo, S. H.; Jun, S. J. Phys. Chem. B 1999, 103, 7743.

volumes, and narrow pore size distributions. As many applications involving MSs exploit the presence of macromolecules confined within the nanopores, an understanding of the infiltration and conformation of macromolecules in nanopores is essential for further advances to be realized. Several theoretical models based on reptation dynamics24,25 have been developed to describe the movement of polymers through confined spaces.26,27 Although these models may help to explain the diffusion of macromolecules within MSs, the infiltration of macromolecules into MSs and the conformation adopted by macromolecules in the nanopores are still poorly understood. Recently, we examined the infiltration of poly(acrylic acid) (PAA) into various amine-functionalized MS particles.28 We reported that the amount of PAA loaded into the particles strongly depends upon the size of the nanopores, the weight-average molecular weight (Mw) of the PAA molecules, and the solution conditions (i.e., pH and ionic strength) employed during the adsorption process. We have also recently demonstrated that MS particles can be used as templates for the synthesis of a novel class of nanoporous polymer spheres (NPSs).29-31 The preparation of NPSs involves the sequential assembly of interacting polymers within the nanopores of MS particles followed by an optional cross-linking step and removal of the particle templates. For example, for the weak polyelectrolyte (PE) pair, PAA and poly(allylamine (22) Goltner, C. G.; Henke, S.; Weissenberger, M. C.; Antonietti, M. Angew. Chem., Int. Ed. Engl. 1998, 37, 613. (23) Kageyama, K.; Tamazawa, J.-I.; Aida, A. Science 1999, 285, 2113. (24) de Gennes, P. G. J. Chem. Phys. 1971, 55, 572. (25) Doi, M.; Edwards, S. F. J. Chem. Soc., Faraday Trans. 2 1978, 74, 1789. (26) Lumpkin, O.; Levene, S. D.; Zimm, B. H. Phys. ReV. A 1989, 39, 6557. (27) Gabashvili, I. S.; Grosberg, A. Y. J. Biomol. Struct. Dyn. 1992, 9, 911. (28) Wang, Y.; Angelatos, A. S.; Dunstan, D. E.; Caruso, F. Macromolecules 2007, 40, 7594. (29) Wang, Y.; Yu, A.; Caruso, F. Angew. Chem., Int. Ed. 2005, 44, 2888. (30) Wang, Y.; Caruso, F. AdV. Mater. 2006, 18, 795. (31) Wang, Y.; Caruso, F. Chem. Mater. 2006, 18, 4089.

10.1021/la703647y CCC: $40.75 © 2008 American Chemical Society Published on Web 03/05/2008

Polyelectrolyte Conformation in Nanopores

hydrochloride) (PAH),29 PAA is first deposited within the nanopores of amine-functionalized MS particles, after which chemical or thermal cross-linking is performed to selectively form amide bonds between the carboxylic acid groups of the PAA and the primary amine groups grafted onto the MS particles. This cross-linking step stabilizes the adsorbed PAA molecules. PAH is then deposited, followed by cross-linking to bond the primary amine groups of the PAH with the carboxylic acid groups of the PAA. Removal of the MS particle template with dilute hydrofluoric acid results in PAA/PAH NPSs. Cross-linking is an important requirement in the preparation of these NPSs, which can be formed when two or more PE layers are deposited within the nanopores. In stark contrast, when the same PAA/PAH system is deposited on nonporous particles, stable capsules are produced (following removal of the templating particles) without the need for cross-linking between the PE layers.32,33 The necessity for cross-linking in the synthesis of PAA/PAH NPSs suggests that the PEs adopt a different conformation in the nanopores of the MS particles than when on the surface of the nonporous particles. The confined geometry associated with the nanopores may cause the PEs to adopt a more coiled conformation once adsorbed, resulting in fewer sites for electrostatic association between the oppositely charged PE layers deposited within the MS particles, and hence the need for cross-linking to stabilize the layers. This finding, coupled with our interest in understanding the conformation of macromolecules (e.g., PEs, peptides, proteins) in nanopores, which is important to further advance the application of MS materials, forms the motivation for the work reported herein. The present study examines the conformation of the weak polycation, PAH, in negatively charged bimodal MS (BMS) particles. PAH was selected since it has a strong affinity for 1,3,6,8-pyrenetetrasulfonic acid tetrasodium salt (4-PSA), an anionic dye used to probe the conformation of PAH due to its unique fluorescent properties. At low concentrations in pure water, 4-PSA exhibits monomer emission only (i.e., maxima at 384 and 403 nm and a shoulder at 424 nm) since the distance between the 4-PSA molecules free in solution is relatively large.34 In the presence of low concentrations of PAH, however, excimer emission is predominantly observed (i.e., a broad, structureless band centered around 500 nm) because the distance between the 4-PSA molecules is greatly reduced upon binding to the charged groups along the PAH chains.34 The critical distance for excimer formation between two pyrene moieties is estimated to be ca. 3-4.5 Å.35,36 Measurement of the ratio of the excimer emission intensity (IE) to the monomer emission intensity (IM) can provide valuable insights into the relative proximity of pyrene molecules and hence the conformation of the macromolecules with which they are associated. For example, IE/IM values approaching zero imply that the distance between the pyrene molecules is significant, and hence the macromolecules are relatively linear, whereas IE/IM values approaching infinity suggest that the distance between the pyrene molecules is small, and hence the macromolecules are more coiled. IE/IM measurements have been used to examine the effect of PE flexibility and binding site topology on the electrostatic binding of 4-PSA molecules in solution34 and to elucidate the nature of the electrostatic interactions between (32) Kato, N.; Schuetz, P.; Fery, A.; Caruso, F. Macromolecules 2002, 35, 9780. (33) Gao, C. Y.; Mo¨hwald, H.; Shen, J. C. AdV. Mater. 2003, 15, 930. (34) Caruso, F.; Donath, E.; Mo¨hwald, H.; Georgieva, R. Macromolecules 1998, 31, 7365. (35) Sisido, M. Prog. Polym. Sci. 1992, 17, 699. (36) Reynisson, J.; Vejby-Christensen, L.; Wilbrandt, R.; Harrit, N.; Berg, R. H. J. Pept. Sci. 2000, 6, 603.

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PEs in ultrathin multilayer films.37 More recently, Park and Hammond have used IE/IM measurements to follow the conformational changes in pyrene-labeled PAH upon adsorption onto neutral hydrophobic surfaces and after the assembly of PE multilayers on adsorbed pyrene-labeled PAH molecules.38 Herein, the infiltration and conformation of PAH in BMS particles are studied as a function of various PAH adsorption conditions (i.e., time, Mw, sodium chloride (NaCl) concentration, and pH). The degree of PAH infiltration into the BMS particles was quantified through thermogravimetric analysis (TGA), and the conformation of the PAH molecules in the nanopores was probed by incubating the PAH-loaded BMS (BMSPAH) particles in 4-PSA and measuring IE/IM of the PAH/4-PSA-loaded BMS (BMSPAH/4-PSA) particles via fluorescence spectroscopy. These experiments were repeated using nonporous silica (NS) particles to demonstrate that the nanopores within the BMS particles significantly influence the extent of PAH adsorption and the conformation of the adsorbed PAH molecules. Confocal laser scanning microscopy (CLSM) was employed to visualize PAH adsorption for the BMS and NS particles. Finally, the PAH/4PSA stoichiometry at saturation binding was estimated through titration experiments. Experimental Section Materials. The BMS particles (particle diameter, 2-4 µm; pore diameters, 2-3 nm and 10-40 nm) were synthesized via the protocol reported by Schulz-Ekloff et al.39 Our previous work has shown that PE adsorption occurs predominantly in the 10-40 nm pores (volume, 1.2 mL g-1).29 The NS particles (diameter, 3 µm) and the 4-PSA were obtained from microParticles GmbH (Berlin, Germany) and Invitrogen (Victoria, Australia), respectively. The water used in all experiments was prepared in a three-stage Millipore Milli-Q Plus 185 purification system and had a resistivity greater than 18 MΩ cm. All other materials were purchased from Sigma-Aldrich and used as received. Methods. Infiltration of PAH into BMS Particles. The BMSPAH particles for TGA were prepared by adding 2 mL of an aqueous 5 mg mL-1 PAH solution (Mw, 15 or 70 kDa; NaCl concentration, 0-2 M; pH, 2-11) to 10 mg of BMS powder (PAH:silica mass ratio, 1:1). The particles were incubated with continual mixing (adsorption time, 0-24 h) and then washed via four cycles of centrifugation (1000g, 30 s)/supernatant exchange (with water)/ redispersion, after which the particles were left to dry at room temperature overnight. Conformation of PAH in BMS Particles. The BMSPAH/4-PSA particles for fluorescence spectroscopy were prepared by adding 0.2 mL of an aqueous 5 mg mL-1 PAH solution (Mw, 15 or 70 kDa; NaCl concentration, 0-2 M; pH, 2-11) to 1 mg of BMS powder (PAH:silica mass ratio, 1:1). The particles were incubated with continual mixing (adsorption time, 0-24 h) and then washed via four cycles of centrifugation (1000g, 30 s)/supernatant exchange (with water)/redispersion. The particles were then incubated for 1 h with continual mixing in 0.2 mL of an aqueous 1 mM 4-PSA solution (pH, 7), after which the particles were washed via four cycles of centrifugation (1000g, 30 s)/supernatant exchange (with water)/redispersion. The 4-PSA concentration used is sufficient to saturate the binding sites of the adsorbed PAH molecules. Control Experiments. As a control, the above experiments were repeated using NS particles. In the TGA experiments, 200 µL of an aqueous 50 mg mL-1 NS particle solution was used instead of 10 mg of BMS powder, and in the fluorescence spectroscopy experiments, 20 µL of an aqueous 50 mg mL-1 NS particle solution was used instead of 1 mg of BMS powder. (37) Caruso, F.; Lichtenfeld, H.; Donath, E.; Mo¨hwald, H. Macromolecules 1999, 32, 2317. (38) Park, J.; Hammond, P. T. Macromolecules 2005, 38, 10542. (39) Schulz-Ekloff, G.; Rathousky´, J.; Zukal, A. Int. J. Inorg. Mater. 1999, 1, 97.

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Figure 1. Fluorescence emission spectra of 4-PSA: (a) monomer emission (IE/IM f 0); (b) excimer emission (IE/IM f ∞); and (c) monomer and excimer emissions (0 < IE/IM < ∞). The inset shows the chemical structure of 4-PSA. PAH/4-PSA Stoichiometry at Saturation Binding. The BMSPAH particles used to estimate the PAH/4-PSA stoichiometry at saturation binding were prepared as described above (PAH adsorption conditions: time, 24 h; Mw, 70 kDa; NaCl concentration, 0.5 M; and pH, 8). A sample portion of the BMSPAH particles (0.1 mg particles in 0.5 mL of water) was gradually titrated with small volumes of an aqueous 1 mM 4-PSA solution (pH, 7) across a 4-PSA concentration range of 5-200 µM. A fluorescence emission spectrum was recorded immediately after each 4-PSA addition. Instrumentation. TGA experiments were conducted on a Mettler Toledo/TGA/SDTA851e Module analyzer. The samples were heated from 25 to 120 °C with a heating rate of 5 °C/min and kept at 120 °C for 20 min under nitrogen (30 mL min-1). They were then heated from 120 to 550 °C with a heating rate of 10 °C/min under oxygen (30 mL min-1). 4-PSA fluorescence emission spectra were recorded on a HORIBA Jobin Yvon Fluorolog using a quartz cuvette (sample volume, 0.2 mL): excitation wavelength, 350 nm; emission wavelength range, 360-600 nm; increment size, 1 nm; excitation and emission slit widths, 1 nm; and integration time, 0.5 s. The BMSPAH/4-PSA particles and the PAH/4-PSA-coated NS (NSPAH/4-PSA) particles were imaged using a Leica TCS SP2 AOBS CLSM. The samples (1 µL) were placed onto glass coverslips and viewed using a 63 × oil immersion objective.

Results and Discussion Infiltration and Conformation of PAH in BMS Particles. The infiltration and conformation of PAH in BMS particles were studied as a function of various PAH adsorption conditions, such as time (0-24 h), Mw (15 and 70 kDa), NaCl concentration (0-2 M), and pH (2-11). The same experiments were conducted using NS particles to determine the extent to which the nanopores within the BMS particles influence the degree of PAH adsorption and the conformation of the adsorbed PAH molecules. TGA was employed to quantify PAH adsorption for the BMS and NS particles under the various PAH adsorption conditions. To investigate the effect of the different PAH adsorption conditions on the conformation of the adsorbed PAH molecules, the BMSPAH and PAH-coated NS particles were incubated in 4-PSA and fluorescence spectroscopy was used to measure IE/IM of the resulting BMSPAH/4-PSA and NSPAH/4-PSA particles. The different types of 4-PSA fluorescence emission spectra obtained are shown in Figure 1: monomer emission only (IE/IM f 0) (Figure 1a), excimer emission only (IE/IM f ∞) (Figure 1b), and monomer

Figure 2. Influence of PAH adsorption time on: (a) PAH adsorption for the BMS and NS particles; and (b) IE/IM of the BMSPAH/4-PSA and NSPAH/4-PSA particles. Fixed PAH adsorption conditions: Mw, 70 kDa; NaCl concentration, 0.5 M; and pH, 8.

and excimer emissions (0 < IE/IM < ∞) (Figure 1c). No fluorescence emission was observed from BMS and NS particles incubated in 4-PSA alone, which indicates that 4-PSA does not bind to silica particles in the absence of preadsorbed PAH. Influence of PAH Adsorption Time. Figure 2a illustrates the variation in PAH adsorption with time for the BMS and NS particles. The infiltration of PAH into the BMS particles increases significantly within the first 45 min and then continues to rise at a considerably slower rate before reaching saturation (0.13 mg PAH per mg silica) after 12 h. In our previous study,28 saturation loading of PAA (Mw, 30 kDa) into amine-functionalized MS particles with ordered nanopores (pore diameter, 6 nm) was achieved after 4 h under similar adsorption conditions. Possible reasons for this faster loading rate are: (i) the 30 kDa PAA may access the nanopores more readily than the 70 kDa PAH, owing to its smaller size; and (ii) the ordered hexagonal arrangement of the 6 nm pores, as opposed to the random arrangement of the nanopores within the BMS particles, may create a less tortuous path, thus facilitating PE infiltration. In the case of the NS particles, saturation coverage (ca. 5 ng PAH per mg silica) occurred within the first 45 min of PAH adsorption (Figure 2a). Comparison of the TGA results for the BMS and NS particles indicates that the nanopores within the

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Figure 3. CLSM images of: (a) BMSPAH/4-PSA particles; and (b) NSPAH/4-PSA particles. PAH adsorption conditions: time, 24 h; Mw, 70 kDa; NaCl concentration, 0.5 M; and pH, 8. Table 1. Influence of PAH Mw on PAH Adsorption for the BMS and NS Particles, and IE/IM of the BMSPAH/4-PSA and NSPAH/4-PSA Particlesa PAH adsorption (mg PAH/mg silica)

normalized IE/IMb

Mw (kDa)

BMS

NS

BMSPAH/4-PSA

NSPAH/4-PSA

15 70

0.15 0.13

7.0 × 10-3 6.0 × 10-3

0.82 1.0

6.2 × 10-4 1.4 × 10-3

a PAH adsorption conditions: time, 24 h; NaCl concentration, 0.5 M; and pH, 8. b The IE/IM of 70 kDa PAH-loaded BMS spheres was set as 1.0.

BMS particles provide significantly more area for adsorption than the outer surface of the NS particles, since the level of PAH adsorption at saturation is significantly greater for the BMS particles. However, the surface associated with the NS particles is more readily accessible to the adsorbing PAH molecules than that associated with the BMS particles, since the rate at which saturation is attained is considerably faster for the NS particles. CLSM was employed to confirm the TGA data obtained, that is, PAH adsorption (and hence the 4-PSA fluorescence) is uniformly distributed across the BMS particles because they contain nanopores (Figure 3a), whereas PAH adsorption (and hence the 4-PSA fluorescence) is confined to the outer surface of the NS particles (Figure 3b). Figure 2b shows the trend in IE/IM of the BMSPAH/4-PSA and NSPAH/4-PSA particles with PAH adsorption time. IE/IM of the BMSPAH/4-PSA particles increases sharply within the first 90 min and then remains essentially constant until 6 h, after which there is a slight decrease in IE/IM. A possible explanation for this result is that, initially, as the PAH adsorption time increases (