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Colloidal Crystals of Silica-Homopolypeptide Composite Particles
Scheme 1. Production of Silica-Polypeptide Colloidal Composite Particles, Not Drawn to Scale
Brian Fong,† Sibel Turksen, Paul S. Russo,* and Wieslaw Stryjewski Department of Chemistry and Macromolecular Studies Group, Louisiana State University, Baton Rouge, Louisiana 70803 Received May 4, 2003. In Final Form: October 2, 2003
Introduction Colloidal particles of uniform size can crystallize in suspension. The large lattice spacing of these crystals leads to diffraction of visible light. Such systems have been considered as optical filters, intensity limiters, photonic band gap materials, and sensors.1-6 Crystallizable colloidal particles are also useful model systems for studying orderdisorder transitions and crystal growth rate.7-9 The most popular formulations are polymer latex or silica. A few years ago, this laboratory described core-shell colloids in which a silica core was coated with poly(γ-benzyl-Lglutamate), PBLG, a helical, rodlike homopolypeptide.10 Compared to previous polypeptide-coated particles,11,12 excellent, latex-like particle uniformity was achieved. The shells of those particles were largely in the R-helical conformation. The following factors motivate continued interest in silica core/homopolypeptide shell composite particles: the helix-coil transition may lead to a responsive, configurable surface; polypeptides display a very rich chemistry; tethering polypeptides to a rigid core may alter their tendency to form cholesteric liquid crystals; colloids with a chiral surface may be useful for some separations. In this short contribution, we describe silica colloidal particles coated with a different homopolypeptide, poly(-carbobenzyloxy-L-lysine) or PCBL. The resultant coreshell particles display crystalline order not yet observed in the PBLG-coated spheres. Materials and Methods Synthesis of the N-Carboxy Anhydride (NCA) of N6Carbobenzoxy-L-lysine. Typically, 5 g (0.018 mol) of the N6carbobenzoxy-L-lysine (CBL) was added to a dry Schlenk flask * Corresponding author (
[email protected]). † Present address: Buckeye Technologies Inc., 1001 Tillman, PO Box 80407, Memphis, TN 38108. (1) Hiltner, P. A.; Krieger, I. M. J. Phys. Chem. 1969, 73 (7), 23862389. (2) Asher, S. A.; Chang, S. Y.; Tse, A. S.; Liu, L.; Pan, G.; Wu, Z.; Li, P. Mater. Res. Symp. Proc. 1995, 374, 305-310. (3) Lee, K.; Asher, S. A. J. Am. Chem. Soc. 2000, 122, 9534-9537. (4) Jethmalani, J. M.; Ford, W. T.; Beaucage, G. Langmuir 1997, 13 (13), 3338-3344. (5) Jethmalani, J. M.; Ford, W. T. Chem. Mater. 1996, 8 (8), 21382146. (6) Eustis, S.; Debord, S. B.; Lofye, M. T.; Lyon, L. A. Adv. Mater. 2002, 14 (9), 658-662. (7) Gast, A. P.; Monovoukas, Y. Nature 1991, 351 (13 June), 553555. (8) Chaikin, P. M.; Pincus, P.; Alexander, S. J. Colloid Interface Sci. 1982, 89 (2), 555-562. (9) He, Y.; Olivier, B.; Ackerson, B. J. Langmuir 1997, 13, 14081412. (10) Fong, B.; Russo, P. S. Langmuir 1999, 15 4421-4426. (11) Tsubokawa, N.; Kobayashi, K.; Sone, Y. Polym. J. 1987, 19 (10), 1147-1155. (12) Dietz, V. E.; Fery, N.; Hamann, K. Angew. Makromol. Chem. 1974, 35, 115-129.
containing 150 mL of ethyl acetate previously distilled over calcium hydride. Following Poche´ and Daly,13 we should add 0.006 mol (1.8 g) of triphosgene to effect the ring closure. For complete conversion of all lysine to the NCA, a slight excess (0.2 g) of the triphosgene was added. The slurry was brought to reflux. After 1.5 h, CBL was incompletely dissolved. Addition of a very small amount of triphosgene improved the dissolution. This was done two more times over the course of the reaction in order to fully react the carbobenzoxy-L-lysine and aid its dissolution. Afterward, a semiclear solution slowly developed. The semiclear solution was refluxed for 1.5 h and then sparged with dry nitrogen until no HCl gas was detected with pH paper from the ethyl acetate vapors. The solution was filtered through Celite diatomaceous earth (Aldrich) into a Schlenk flask. A vacuum was applied to concentrate the NCA eluent. Dry hexane was then poured into the flask, and it was swirled around to initiate crystallization. The crystallized solution was then filtered with a glass-fritted funnel under a flowing blanket of nitrogen. The NCA was then dissolved in minimal THF, and hexane was added to initiate recrystallization. The crystals were filtered again and a proton NMR spectrum was recorded. Typical yields were 60%, corresponding to about 3 g of NCA. Synthesis of the PCBL Composite Colloidal Particles. The NCA was dissolved in 50 mL of THF previously distilled over potassium. Amino-functionalized silica spheres, produced as described previously,10 were dispersed in N,N′-dimethylformamide, DMF, and then injected into the NCA solution. A drying tube with Drierite (anhydrous CaSO4) was attached, and the NCA solution was allowed to stir for 3 days. The composite colloidal solution was centrifuged down to a pellet, which was washed with THF, THF/DMF, and then finally DMF in order to remove any free polymers. Infrared Spectroscopy. Infrared spectra were recorded with a Perkin-Elmer 1760 FT-IR using GRAMS 386 software. Dry samples were prepared as KBr pellets. Nuclear Magnetic Resonance Spectroscopy. A Bruker 200 MHz NMR was used to follow the formation of the NCA ring from the reaction between glutamic acid and triphosgene. Ring formation leads to a slight shift upfield for the hydrogen attached to the nitrogen from 6.9 to 6.5 ppm. Also, 13C NMR shows the presence of three carbonyl carbons on the N-carboxy anhydride monomer. Visible Spectroscopy. As the characteristic domain size of our crystals is small, we used an Olympus BH2 polarized optical microscope to focus on one or several domains. The transmitted light was channeled to a monochromator/detector system (SLM Aminco 8000, SLM Instruments, Inc.) using fiber optic cable. (13) Daly, W. H.; Poche´, D. S. Tetrahedron Lett. 1988, 29 (46), 58595862.
10.1021/la034762u CCC: $27.50 © 2004 American Chemical Society Published on Web 11/21/2003
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Langmuir, Vol. 20, No. 1, 2004 267 grid, which was then coated with carbon by resistance vaporization in a Balzers/Union MED010 deposition system. The grid was attached to an aluminum mounting stub with double-sided tape and conductive paint, sputter coated with gold/palladium in an Edwards 5150 sputter coater, and examined at 15 kV with a Cambridge 260 Stereoscan scanning electron microscope.
Results and Discussion
Figure 1. Apparent hydrodynamic radius for PCBL-coated silica particles at different scattering vectors.
Figure 2. Scanning electron micrograph of PCBL-coated silica particles prepared from DMF.
Figure 3. Infrared spectrum of PCBL-coated silica particles. Dynamic Light Scattering (DLS). Dynamic light scattering was performed on a custom-built apparatus using an ALV-5000 digital autocorrelator. Particles were dispersed in DMF at controlled temperatures ranging from 20 to 30 °C. Cumulants14 and single-exponential analyses were used to fit the data. The hydrodynamic size was obtained as usual from the StokesEinstein equation.15 Electron Microscopy. Approximately 5 µL of the sample dispersion was pipetted onto a collodion-coated copper specimen (14) Koppel, D. E. J. Chem. Phys. 1972, 57, 4814-4820. (15) Tanford, C. Physical Chemistry of Macromolecules; John Wiley & Sons: New York, 1961.
The correlation functions from DLS were nearly perfect single exponentials. Third-order cumulants analysis and single exponential analysis gave effectively the same hydrodynamic radius of Rh ) 2516 ( 14 Å in DMF from 20 to 30 °C based on multiple angle measurements. From previous results10 we expect a core radius of about 970 Å, so the PCBL thickness can be estimated as 1500 Å. There was effectively no temperature trend, independent of solid weight fraction in the range 0.0002-0.0008. A qualitative indicator of particle uniformity is available from the dimensionless ratio of the second cumulant to the square of the first,14 often called µ2/Γ2. This parameter, which would be zero for perfectly uniform particle size, was so low as to defy measurement (the largest value observed in any single measurement was 0.04). Another measure of particle quality is the independence of apparent hydrodynamic radius with angle. In a polydisperse suspension, large particles scatter more weakly to high angles, so that the faster, smaller particles assume greater importance. This leads to a rising Rh vs q2 trend. Nonspherical shape can cause a similar effect when the particles are sufficiently large. As with the earlier, PBLGcoated particles,10 absence of any trend in Figure 1 testifies to the uniformity and spherical symmetry of the particles. A typical scanning electron microscopy (SEM) image, Figure 2, confirms the size uniformity and generally spherical shape. Some of the apparently misshapen particles might assume a rounder form when dispersed in solution. As in ref 10, no attempt was made to size the particles from the SEM images; they appear smaller than when measured in the fully solvated state by DLS. Infrared spectra are often used to characterize the conformation of polypeptides.16-26 Two peaks due to the backbone amides are of particular importance. For R-helices, the amide I peak due to CdO stretching is found near 1650 cm-1 while the amide II peak resulting from C-N stretching and N-H bending is found between 1535 and 1550 cm-1. For β-sheets, the amide I peak is located at a frequency some 30 cm-1 lower. The amide II peak for β-sheets also tends to lie at lower values, but the separation from the R-helix is less. We observed peaks at 1543 and (16) Combelas, Ph.; Garrigou-Lagrange, C. J. Polym. Sci., Part C: Polym. Lett. 1972, 39, 211-218. (17) Doty, P.; Holtzer, A. M.; Bradbury, J. H.; Blout, E. R. J. Am. Chem. Soc. 1954, 76 (September 6), 4493-4494. (18) Sugai, S.; Kamashima, K.; Nitta, K. J. Polym. Sci., Part A-2 1968, 6, 1065-1081. (19) Brumberger, H.; Cheng, B. Biopolymers 1974, 13, 2653-2654. (20) Wieringa, A. M.; Siesling, E. A.; Werkman, P. J.; Angerman, H. J.; Vorenkamp, E. J.; Schouten, A. J. Langmuir 2001, 17 (21), 64856490. (21) Wieringa, A. M.; Siesling, E. A.; Werkman, P. J.; Vorenkamp, E. J.; Schouten, A. J. Langmuir 2001, 17 (21), 6491-6495. (22) Miyazawa, T.; Blout, E. R. J. Am. Chem. Soc. 1961, 83, 712719. (23) Wieringa, A. M.; Siesling, E. A.; Geurts, P. F. M.; Werkman, P. J.; Vorenkamp, E. J.; Erb, V.; Stamm, M.; Schouten, A. J. Langmuir 2001, 17, 6477-6484. (24) Fasman, G. D.; Idelson, M.; Blout, E. R. J. Am. Chem. Soc. 1961, 83 (Feb. 5), 709-712. (25) Parrish, R. P., Jr.; Blout, E. R. Biopolymers 1971, 10, 14911512. (26) Hayashi, T.; Nakajima, A. Bull. Inst. Chem. Res., Kyoto Univ. 1977, 55 (2), 150-162.
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Figure 4. Photomicrographs of colloidal crystals containing PCBL-coated silica particles in N,N′-dimethylformamide, under crossed polars: A, variety of colors due to differing domain orientations; B, detail of bands due to crystal twinning.
1653 cm-1, Figure 3, consistent with a predominance of the R-helix structure. The most striking observation about these particles is their ability to form colloidal crystals, as shown in Figure 4. Monovoukas and Gast27,28 explained the appearance of bright colors when observed between crossed polarizers in a microscope, despite the cubic symmetry of colloidal crystals. The effect arises from polarization-dependent diffraction; effectively, light of a given polarization is diffracted out of the crystal, while light of that same color but different polarization is transmitted. The bands traversing some domains could be made to exchange colors by rotating the sample stage, which indicates crystal twinning.27 If the sample is rotated 90°, the original band colors are restored. While colloidal crystals of latex spheres at low ionic strength are stabilized by charge repulsions, the composite silica-polypeptide are not expected to carry a high charge. The existence of light-diffracting colloidal crystals is understood in terms of packing considerations for particles of an appropriate size. The particles have not yet been produced in amounts that would permit accurate determination of the concentration within the crystal phase; however, similar crystals have been observed in m-cresol dispersions. Crystal formation is slow, typically 3-4 weeks. Centrifugation hastens crystal formation but seems to produce smaller crystals. The delicate crystals are destroyed easily, even by gentle tilting. The size of a typical domain is 0.5 mm. The optical contrast between the particles and solvent is low. While charged latex colloidal crystals in aqueous media often display striking opalescence in natural light, very close inspection is required to see any “sparklers” in the silica-PCBL composite particle suspensions. Low contrast and small domain size may explain why efforts to measure Kossel patterns28 have so far been unsuccessful, and structures have not been revealed in epi-illumination confocal microscopy. In transmission, and between crossed polars, (27) Monovoukas, Y.; Gast, A. P. Langmuir 1991, 7, 460-468. (28) Monovoukas, Y.; Gast, A. P. J. Colloid Interface Sci. 1989, 128 (2), 533-548.
Figure 5. Optical transmission between crossed polars as a function of wavelength. Only a few crystalline domains were observed.
the color purity within any given domain is very good. As shown in Figure 5, transmission bandwidths of about 20 nm are achieved; this is comparable to an interference filter. Conclusion Nearly uniform composite silica-PCBL particles of an appropriate size spontaneously form colloidal crystals. The optical properties seem similar to those of colloidal crystals formed by achiral particles. No evidence of the cholesteric, lyotropic phase seen in PCBL was found; this does not preclude the possibility that such a phase could be observed in denser suspensions (perhaps prepared by strong centrifugal fields) or for particles having different coreshell dimensions. The helix-coil transition available to the PCBL polymer, even in a single solvent,29 may prove useful for annealing the structures into larger single (29) Matsuoka, M.; Norisuye, T.; Teramoto, A.; Fujita, H. Biopolymers 1973, 12, 1515-1532.
Notes
domains, for fine-tuning optical response, or sensing binding of chiral substances. Acknowledgment. We thank Cindy Henk of the Socolofsky Microscopy Facility at LSU for expert assistance with the electron microscope and Dr. Fred Enright of the LSU Department of Veterinary Sciences for making
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available the monochromator. Drew S. Poche and Ioan I. Negulescu of LSU made helpful suggestions or provided instrument assistance. This work was supported by the National Science Foundation (DMR-0075810) and by the donors of the Petroleum Research Fund, administered by the American Chemical Society. LA034762U
