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Self-Assembly of Avidin and D-Biotin-Tethering Zeolite Microcrystals into Fibrous Aggregates Soong Ho Um,† Goo Soo Lee,‡ Yun-Jo Lee,‡ Kee-Kahb Koo,† Chongmok Lee,§ and Kyung Byung Yoon*,‡ Center for Microcrystal Assembly and Departments of Chemistry and Chemical Engineering, Sogang University, Seoul 121-742, Korea, and Department of Chemistry, Ewha Womans University, Seoul 120-750, Korea Received February 4, 2002. In Final Form: February 20, 2002 Avidin and nanometer-sized zeolite crystals (∼300 nm) tethered with D-biotin readily self-assemble into thin (2-20 µm) and very long (>1 cm) fibrous aggregates in a buffer solution of pH ) 7.4 when the avidin-to-zeolite weight ratio (A/Z) is equal to or higher than 0.2. At A/Z ) 0.2, the exteriors of the produced fibrils are covered with zeolite-A crystals. At A/Z ) 0.4, the zeolite crystals are completely buried within the fibrils covered with thick layers of avidin. At A/Z ) 0.8-1.0, the morphology of the fibrils becomes smooth and flat due to the thick surface-lining avidin layers. The zeolite and avidin remain intact within the fibrils. Control experiments reveal that complexation between avidin and the zeolite-bound biotin is essential for fibrillation to occur. Interestingly, discrete clusters of zeolite crystals with sizes of 5-10 µm are produced when the A/Z ratio is reduced to 0.1. The observed fibrillation is very much like the previously reported case with β-glucosidase and D-glucose-tethering zeolite microcrystals, despite that the type of the protein-substrate pair is different. This result therefore raises the possibility that self-assembly of complexforming proteins and the nanoparticles or microparticles tethered with the corresponding substrates into fibrils is a general phenomenon.
Introduction Self-assembly is a process in which atoms, molecules, and systems of molecules spontaneously arrange themselves into functioning structured entities without human intervention, and the most elaborate expression of such processes is demonstrated in life systems.1,2 Great efforts have been directed to gain insights into such an astonishing feat of nature, which are necessary to realize the scientists’ dream to reproduce such processes in the laboratory.1-15 Along these lines, self-assembly of building * To whom correspondence should be addressed. † Department of Chemical Engineering, Sogang University. ‡ Department of Chemistry, Sogang University. § Ewha Womans University. (1) (a) Whitesides, G. M. Sci. Am. 1995, 273, 146-149. (b) Bowden, N. B.; Weck, M.; Choi, I. S.; Whitesides, G. M. Acc. Chem. Res. 2001, 34, 231-238. (2) Ozin, G. A. Chem. Commun. 2000, 419-432. (3) Boal, A. K.; Ilhan, F.; DeRouchey, J. E.; Thurn-Albrecht, T.; Russel, T. P.; Rotello, V. M. Nature 2000, 404, 746-748. (4) Shenton, W.; Davis, S. A.; Mann, S. Adv. Mater. 1999, 11, 449451. (5) Li, M.; Wong, K. K. W.; Mann, S. Chem. Mater. 1999, 11, 23-26. (6) Alivisatos, A. P.; Johnsson, K. P.; Peng, X.; Wilson, T. E.; Loweth, C. J.; Bruchez, Jr M. P.; Schultz, P. G. Nature 1996, 382, 609-611. (7) (a) Taton, T. A.; Mucic, R. C.; Mirkin, C. A.; Letsinger, R. L. J. Am. Chem. Soc. 2000, 122, 6305-6306. (b) Storhoff, J. J.; Mirkin, C. A. Chem. Rev. 1999, 99, 1849-1862. (c) Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. Nature 1996, 382, 607-609. (d) Elghanian, R.; Storhoff, J. J.; Mucic, R. C.; Letsinger, R. L.; Mirkin, C. A. Science 1997, 277, 1078-1081. (8) Dujardin, E.; Hsin, L-.B.; Wang, C. R. C.; Mann, S. Chem. Commun. 2001, 1264-1265. (9) Li, M.; Schnablegger, H.; Mann, S. Nature 1999, 402, 393-395. (10) Murray, C. B.; Kagan, C. R.; Bawendi, M. G. Science 1995, 270, 1335-1338. (11) Andres, R. P.; Bielefeld, J. D.; Henderson, J. I.; Janes, D. B.; Kolagunta, V. R.; Kublak, C. P.; Mahoney, W. J.; Osifchin, R. G. Science 1996, 273, 1690-1693. (12) Sun, S.; Murray, C. B.; Weller, D.; Folks, L.; Moser, A. Science 2000, 287, 1989-1992. (13) Stupp, S. I.; LeBonheur, V.; Walker, K.; Li, L. S.; Huggins, K. E.; Keser, M.; Amstutz, A. Science 1997, 276, 384-389. (14) Pileni, M. P. J. Phys. Chem. B 2001, 105, 3358-3371.
blocks through biologically important interactions such as DNA base pairing, antigen-antibody interaction, and the related avidin-biotin complexation has received special attention as a means to gain insights into the natural biological self-assembly processes.1-8 Recently, stemming from our interests in the mono- and multilayer assembly of zeolite microcrystals on supports,16 we discovered a novel phenomenon that β-glucosidase and the D-glucose-tethering zeolite crystals readily self-assemble into thin (2-20 µm) and long fibrils (>1 cm) upon stirring in aqueous solution.17 The zeolite crystals and β-glucosidase remain intact within the fibrils, and the enzymatic activity of β-glucosidase in the fibrils is also preserved even after they were stored in water for 6 months at room temperature. The above fibrillation phenomenon was proposed to occur through complexation of β-glucosidase with the D-glucose moieties tethered to the zeolite surfaces followed by crystallization of the enzyme on top of the surface-coating enzyme layer to such an extent that the inorganic crystals are interconnected by the crystalline enzyme. Although the mechanism for fibrillation still remains unclear, it is necessary to examine the generality of the above intriguing phenomenon by testing other pairs of complex-forming proteins and nanometer- or micrometer-sized inert supports tethered with the correspond(15) Brust, M.; Bethell, D.; Schiffrin, D. J.; Kiely, C. J. Adv. Mater. 1995, 7, 795-797. (16) (a) Kulak, A.; Lee, Y.-J.; Park, Y. S.; Yoon, K. B. Angew. Chem., Int. Ed. 2000, 39, 950-953. (b) Choi, S. Y.; Lee, Y.-J.; Park, Y. S.; Ha, K.; Yoon, K. B. J. Am. Chem. Soc. 2000, 122, 5201-5209. (c) Ha, K.; Lee, Y.-J.; Lee, H. J.; Yoon, K. B. Adv. Mater. 2000, 12. 1114-1117. (d) Ha, K.; Lee, Y.-J.; Jung, D.-Y.; Lee, J. H. Yoon, K. B. Adv. Mater. 2000, 12, 1614-1617. (e) Kulak, A.; Park, Y. S.; Lee, Y.-J.; Chun, Y. S.; Ha, K.; Yoon, K. B. J. Am. Chem. Soc. 2000, 122, 9308-9309. (f) Lee, G. S.; Lee, Y.-J.; Ha, K.; Yoon, K. B. Tetrahedron 2000, 56, 6965-6968. (g) Ha, K.; Lee, Y.-J.; Chun, Y. S.; Park, Y. S.; Lee, G. S.; Yoon, K. B. Adv. Mater. 2001, 13, 594-596. (h) Lee, G. S.; Lee, Y.-J.; Yoon, K. B. J. Am. Chem. Soc. 2001, 123, 9769-9779. (i) Lee, G. S.; Lee, Y.-J.; Ha, K.; Yoon, K. B. Adv. Mater. 2001, 13, 1491-1495. (17) Lee, G. S.; Lee, Y.-J.; Choi, S. Y.; Park, Y. S.; Yoon, K. B. J. Am. Chem. Soc. 2000, 122, 12151-12157.
10.1021/la020118o CCC: $22.00 © 2002 American Chemical Society Published on Web 04/11/2002
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Langmuir, Vol. 18, No. 11, 2002 Scheme 1
ing substrates. As a test case, we have examined avidin and D-biotin as an alternative protein-substrate pair, which is known to form a strong complex.18 We now report that even the newly tested protein-substrate pair is very much like that of β-glucosidase and D-glucose in terms of fibrillation, despite the fact that the complex-forming protein-substrate systems are different from the molecular respects. Experimental Section Materials. Avidin (lot number 38H7834), D-biotin (lot number 129H0974), and phosphate buffered saline (PBS, pH ) 7.4) were purchased from Sigma, and they were used as received. 1,3Dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS), 4-(N,N-dimethylamino)pyridine (DMAP), and hydrogen hexachloroplatinate(IV) (H2PtCl6 ‚xH2O) were purchased from Aldrich and used as received. Trimethoxysilane from Aldrich was distilled and kept in a Schlenk storage flask under argon. N,N-Dimethylformamide (DMF) from Junsei was first dried with activated granular Linde 4A prior to vacuum distillation. The distilled DMF was stored in a Schlenk storage flask in the dark. Toluene from Junsei was treated with concentrated sulfuric acid until the sulfuric acid layer remains colorless, and the water-washed toluene was dried with anhydrous calcium chloride prior to distillation over sodium under argon. The distilled toluene was transferred to a Schlenk storage flask with the help of a Teflon cannular under counter flow of argon. Zeolite-A (Na+ form) was synthesized according to the literature procedure using tetramethylammonium (TMA+) as the template.19 The average size of the resulting zeolite-A was ∼300 nm. TMA+ was removed from the zeolite by calcining it at 550 °C for 12 h prior to biotin tethering on their external surfaces. Preparation of N-Hydroxysuccinimidyl Biotin Ester (1) (Step I in Scheme 1). D-Biotin (1 g, 4.09 mmol) was first dissolved in hot DMF (25 mL, 80 °C), and the solution was cooled to room temperature. Subsequently, NHS (0.49 g, 4.26 mmol) and DCC (0.84 g, 4.07 mmol) were added into the solution. After the mixture was stirred for 2 h, the precipitated dicyclohexylurea was removed by filtration. The volume of the filtered solution was reduced to ∼10 mL by applying vacuum at 50 °C, and diethyl ether (100 mL) was added into the residual solution to precipitate the product 1. The filtered product was washed successively with diethyl ether (100 mL) and 2-propanol (100 mL). C10H19N3O5S: 341.39, yield 0.98 g (70%): white solid; 1H NMR (DMSO, δ/ppm) 6.7 (bs, 1H, NH), 4.3 (m, 2H, NCHCHN), 3.15 (bd, 1H, CHS), 2.81 (s, 4H, COCH2CH2CO), 2.75 (m, 2H, CH2S), 2.22 (t, 2H, CH2), 1.5 (bm, 6H). Preparation of Undecylenyl Biotin Ester (2) (Step II in Scheme 1). Compound 1 (100 mg, 0.29 mmol), ω-undecylenyl (18) The dissociation constant for the avidin-biotin complex is ∼10-15 M. See: Hiller, Y.; Gershoni, J. M.; Bayer, E. A.; Wilchek, M. Biochem. J. 1987, 248, 167-171. (19) Zhu, G.; Qui, S.; Yu, J.; Sakamoto, Y.; Xiao, F.; Xu, R.; Terasaki, O. Chem. Mater. 1998, 10, 1483-1486.
Um et al. Scheme 2
alcohol (49 mg, 0.29 mmol), and DMAP (3.6 mg, 0.29 mmol) were dissolved in anhydrous DMF (25 mL) at room temperature, and the solution was stirred at 50 °C for 48 h. Silica gel (1 g) was added into the solution, and the solvent was removed by applying vacuum. Compound 2 impregnated onto silica gel was purified by flash chromatography with the mixture of methanol and dichloromethane (6:94) as the eluant. C21H36N2O3S: 396.61; yield 10 mg (10%); white solid; mp 105-107 °C; 1H NMR (CDCl3, δ/ppm) 5.8 (m, 1H, CH), 5.2 (s, 1H, NH), 4.95 (m, 2H, CH2), 4.9 (s, 1H, NH), 4.5 (m, 1H, NCH), 4.3 (m, 1H, NCH), 4.1 (t, 2H, COOCH2), 3.2 (bd, 1H, CHS), 2.96 (m, 1H, CH2S), 2.75 (bs, 1H, CH2S), 2.35 (t, 2H, CH2COO), 2.0 (m, 2H, CH2CHCH2), 1.5 (bm, 22H). Preparation of Biotinylated Undecyltrimethoxysilane (Biotin-UD-TMS) (Step III in Scheme 1). Anhydrous toluene (5 mL) and trimethoxysilane (0.08 mL, 78 mg, 0.64 mmol) were successively introduced into a Schlenk flask containing both 2 (50 mg, 0.13 mmol) and H2PtCl6 ‚xH2O (10 mg) using hypodermic syringes under a counterflow of argon, and the mixture was stirred for 16 h at 60 °C. After cooling to room temperature, the mixture was rapidly filtered through packed cotton fibers by applying argon pressure. Biotin-UD-TMS was obtained as a colorless solid by removal of the solvent under vacuum. C24H46N2O6SSi: 518; yield 43.8 mg (67%); slowly decomposes at 180 °C; 1H NMR (CDCl3, δ/ppm) 4.9 (s, 1H, NH), 4.7 (s, 1H, NH), 4.5 (m, 1H, NCH), 4.3 (m, 1H, NCH), 4.1 (t, 2H, COOCH2), 3.6 (s, 9H, CH3O), 3.2 (bd, 1H, CHS), 2.96 (m, 1H, CH2S), 2.75 (bs, 1H, CH2S), 2.35 (t, 2H, CH2COO), 1.5 (m, 24H), 0.9 (t, 2H, CH2Si). Preparation of Undecyltrimethoxysilane (UD-TMS). UDTMS was similarly prepared from 1-undecene and trimethoxysilane. C14H32O3Si: 276.49; yield 36.29 mg (90%); 1H NMR (CDCl3, δ/ppm) 3.6 [s, 9H, (CH3O)3Si], 1.2 (m, 21H), 0.6 (t, 2H, CH2Si). Preparation of Zeolite-A Crystals Tethered with Both Biotin-UD and UD (Biotin-Z) (Scheme 2). Dry toluene (50 mL) was introduced into a Schlenk flask containing freshly dried zeolite-A (100 mg) under a counterflow of argon. The toluene slurry was sonicated for 10 min to disperse the zeolite crystals into the solution. Independently, a toluene solution of biotinUD-TMS (10 mM) (solution A) was prepared by introduction of dry toluene (8.7 mL) into a Schlenk flask containing biotin-UDTMS (45 mg, 0.087 mmol). Independently, a toluene solution of UD-TMS (200 mM) (solution B) was prepared by introduction of 50 mL of toluene into a Schlenk flask containing 2.765 g (10 mmol) of UD-TMS under argon. Solution A (1 mL) and solution B (0.5 mL) were successively transferred to the toluene solution dispersed with zeolite-A (10 mg) under argon, and the mixture was refluxed for 1 h. Consequently, the resulting ratio of biotinUD-TMS and UD-TMS was 1:10 in the solution. After cooling to room temperature, the biotin-tethering zeolite powders (biotinZ) were collected by rapid filtration in the atmosphere over a filter paper. The filtered powders were washed successively with fresh toluene (100 mL) and ethanol (100 mL). The collected powders were dried in an oven at 120 °C for 30 min. Formation of Fibrous Avidin-Biotin-Z Composite Material. To induce fibrillation with avidin, a PBS buffer solution (20 mL)20 dispersed with biotin-Z (10 mg) was introduced into a flat-bottomed cylindrical reaction vessel in which a Teflon slab (20) Avidin is known to be most active at pH 7.4. See: Nelson, K. E.; Gamble, L.; Jung, L. S.; Boeckl, M. S.; Naeemi, E.; Golledge, S. L.; Sasaki, T.; Castner, D. G.; Campbell, C. T.; Stayton, P. S. Langmuir 2001, 17, 2807-2816.
Self-Assembly of Avidin Tethered with Biotin
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Figure 1. Diffuse-reflectance FT-IR spectra of solid biotinUD-TMS (A) and biotin-Z (B). with four supporting legs was placed. Avidin (2 mg, 3 µmol) was subsequently introduced into the solution, and the heterogeneous mixture was stirred with the aid of an underlying small magnetic stirrer at room temperature. The turbid solution slowly turned transparent with time, and after an elapse of 1 day the presence of fibrils on top of the Teflon slab became visually apparent. The fibrils were collected by centrifugation, and the supernatant solution was removed by decantation. Fresh distilled deionized water (10 mL) was introduced into the centrifuge tube containing the coagulated fibrils, and they were redispersed into the freshwater by applying sonication. After shaking for a few minutes, the fibrils were collected again by centrifugation and such a washing cycle was repeated five times. The fibrils were then finally dispersed into 10 mL of freshwater, and the solution was freeze-dried. The amount of avidin was also varied to 1, 4, 8, and 10 mg, respectively, to test the effect of avidin-to-zeolite (A/Z) weight ratio on the morphology of the resulting avidinbiotin-Z composite. Instead of a PBS buffer solution, distilled deionized water was also tested as the solvent. Instrumentation. The scanning electron microscope (SEM) images of zeolites, fibrils, and the lyophilized avidin powder were obtained from a FE-SEM (Hitachi S-4300) at an acceleration voltage of 15 kV. On top of the samples platinum/palladium alloy (in the ratio of 8 to 2) was deposited with a thickness of about 15 nm. The X-ray diffraction patterns were obtained from a Rigaku diffractometer (D/MAX-1C) with the monochromated beam of Cu KR. The diffuse reflectance FT-IR spectra of the samples were obtained from a JASCO FT/IR 620 using a SpectraTech diffuse reflectance accessory. Sonication of the samples was carried out using an ultrasonic cleaning bath operated at 28 kHz.
Results and Discussion I. Confirmation of Attachment of Biotinylated Undecyl Group onto Zeolite Surfaces. The ready attachment of biotinylated undecyl (biotin-UD) moieties onto the zeolite-A surfaces was confirmed by the close matching between the diffuse-reflectance FT-IR spectra of biotin-UD-TMS powders and the zeolite-A crystals treated with the compound (Figure 1). The characteristic carbonyl stretching bands at 1735-1740 and 1765 cm-1 together with the N-H stretching bands in the 33003600 cm-1 region unambiguously confirm the presence of urea moiety21 and hence the biotin moiety on the surfaces (21) Pretsch, E.; Bu¨hlmann, P.; Affolter, C. Structure Determination of Organic Compounds; Springer-Verlag: New York, 2000; Chapter 6.
Figure 2. SEM images showing the A/Z ratio dependent morphology change of the fibrils: 0.2 (A), 0.4 (B), and 1.0 (C). The right part of each panel shows the 10 times magnified image of the rectangular section marked on the left part.
of zeolite crystals. The relatively strong C-H absorption in the 2800-2900 cm-1 region also indicates that there are extra undecyl (UD) groups in addition to biotin-UD moieties on the zeolite surfaces. It was assumed that the average molar ratio of biotin-UD and UD groups attached to the zeolite surface is 1:10 on the basis of the ratio of the initially introduced amounts of biotin-UD-TMS and UDTMS into the reaction mixture. The reason for choosing the molar ratio of 1:10 is to comply with the report by Castner, Stayton, and the co-workers20 that the molar ratio of 1:9, which is close to 1:10, is optimal for effective binding between avidin and the surface-bound biotin. II. Formation of Avidin-Biotin-Z Composites. The PBS buffer solution (pH ) 7.4, 20 mL) dispersed with biotin-UD-tethering zeolite-A (10 mg) remained turbid for an unlimited period of time as long as the solution was kept stirred. However, upon addition of 2 mg of avidin the turbid solution slowly turned transparent with time and after 2 days thin fibrils sedimented on top of the Teflon slab became visually apparent. The yields (∼10 mg) of the isolated (freeze-dried) fibrils were close to the total amounts of biotin-Z (10 mg) and avidin (2 mg). This indicates that the yield is close to quantitative. The typical scanning electron microscopy (SEM) image of the water-washed, freeze-dried fibrils is shown in Figure 2A. As noticed more clearly from the magnified (×10) image shown in the right part, the exteriors of the resulting fibrils are heavily coated with clusters of zeolite crystals due to deficiency of avidin. Upon increase of the A/Z ratio to 0.4, all the zeolite crystals become buried within the interiors of the fibrils that are covered with thick avidin layers, as shown in Figure 2B. Upon further increase of the A/Z ratio to 0.8 or 1.0, the morphology of the fibrils
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Figure 3. SEM image of lyophilized powder of avidin.
becomes smooth and flat, as typically shown in Figure 2C. The above A/Z ratio-dependent change of fibril morphology is very much alike with that of β-glucosidase and D-glucose-tethering zeolite crystals. It is also important to note that the morphologies of the resulting avidinbiotin-Z composite fibrils are different from that of the initially added lyophilized avidin shown in Figure 3, indicating that there are no remaining free avidin molecules which otherwise would lyophilize in the flakelike morphology. The presence of a Teflon support (slab) is not necessary for the above fibrillation to occur. It just helps the fibrils grow longer without being chopped into smaller fragments by being hit by the underlying spinning magnetic bar. Even the magnetic stirring bar is not necessary for the fibrillation to occur since any methods, such as shaking, are equally effective as long as sedimentation of the zeolite particles in the heterogeneous mixture is prevented. The nature of the container (glass or plastic) also does not affect the fibrillation process since the fibrils do not grow on the walls of the containers, indicating that the container walls do not serve as the seeds or anchoring spots for the fibrils to grow. From the fact that the resulting fibrils sediment onto the bottom of the containers, it is inferred that the density of the resulting wet fibrils is larger than that of water. Fibrillation also undergoes in distilled deionized water but at a much slower rate. For instance, while 2 days are usually long enough to complete the fibrillation in PBS solution (pH ) 7.4), the degree of fibrillation proceeds to only ∼50%, even after 10 days of stirring. However, despite the fact that fibrillation is slow in distilled deionized water, the SEM images of the resulting fibrils are almost identical with those images shown in Figure 2, depending on the corresponding A/Z ratio. The above results clearly indicate that the fibrillation rate is much faster at pH ) 7.4 at which the biotin-binding activity of avidin is known to be highest.20 The above result therefore indicates that the activity of avidin is critical for fibrillation, and hence fibrillation proceeds as a result of complexation between avidin and the zeolite-bound biotin moieties. Such fibrillation does not proceed when biotin-free, bare zeolite-A crystals are employed. Also, even the biotintethering zeolite-A does not undergo fibrillation in the PBS solution if addition of avidin is deliberately omitted. Even if avidin is added into the PBS solution dispersed with biotin-Z, the subsequent fibrillation does not proceed if the avidin was thermally denatured by boiling the PBS solution (10 mL) of avidin (4 mg) for 2 h prior to mixing with biotin-Z dispersed in PBS solution (10 mL). Dissolution of a large excess of free D-biotin (244 mg, 1 mmol) into the biotin-Z-dispersed PBS solution prior to addition of fresh avidin also prevents the zeolite and avidin undergoing fibrillation. The above various control experiments unambiguously establish that complexation between avidin and the zeolite-bound D-biotin moieties is the key for fibrillation.
Figure 4. Magic angle spinning solid-state NMR spectra of the fibrils (A/Z ) 0.4) and the corresponding reference materials (as indicated): 27Al (A), 29Si (B), and 13C (C).
One might still suspect that fibrillation is an artifact that arises during freeze-drying. However, it should be kept in mind that the added, highly water-soluble avidin is readily washed away during the repeated (5 times) cycles of centrifugation, decantation of the supernatant solution, and redispersion of collected fibrils into freshwater by sonication. Moreover, slow evaporation of water from the aqueous mixture of fibrils in the atmosphere also leads to isolation of the fibrils. This fact further eliminates the above suspicion. However, the air-drying tends to leave severe wrinkles on the surfaces of fibrils, and this is the reason we prefer freeze-drying, in particular, for SEM analyses. The evidence that the fibrils are composed of avidin and zeolite crystals also comes from the solid-state NMR of the fibrils. For instance, the solid-state NMR (27Al, 29Si, and 13C) of the fibrils obtained from the solution of avidin and biotin-Z with the A/Z ratio of 0.4 revealed not only the presence of the two components but also the fact that they remain intact within the fibrils as shown in Figure 4. Thus, the solid-state NMR spectra of the fibril and the corresponding authentic reference sample for each nucleus of 27Al, 29Si, or 13C are nearly superimposable. Although the presence of zeolite crystals in the fibrils is already apparent for the fibrils with the A/Z ratio of 0.2 from SEM images (Figure 2A), it is not so apparent for fibrils with A/Z g 0.4 (Figure 2B,C) by the SEM images. However the presence of the zeolite-A crystals within the fibrils was also confirmed by the SEM images of the inorganic residues of the fibrils after calcination at 550 °C under flowing oxygen to remove the organic components (avidin). Thus, as shown in Figure 5, the calcined fibrils show aggregates of zeolite crystals in the shapes of fibrils. The SEM images also show the fact that the zeolite crystals
Self-Assembly of Avidin Tethered with Biotin
Figure 5. SEM images showing the aggregates of cubic zeolite-A crystals in the shape of fibrils remained after burning off the fibrils consist of avidin and biotin-Z (A/Z ) 0.4) at 550 °C overnight under flowing oxygen.
are densely packed but rather randomly oriented within the fibrils in addition to the fact that zeolite crystals are indeed present within the fibrils. The above fibrillation phenomenon is very much like that of the previously observed fibrillation of β-glucosidase and D-glucose tethering zeolite-A,17 despite the fact that complex-forming proteins and the corresponding substrates are entirely different from the molecular aspect. This result thus raises the possibility that self-assembly of proteins and the corresponding substrates-tethering inorganic micro- or nanocrystals into fibrils is a general phenomenon. However the present result still does not unveil the reason why axial direction is favored during self-assembly of avidin and biotin-Z and β-glucosidase and D-glucose tethering zeolite-A. As a possible means to investigate the mechanism for fibrillation, we prepared finely ground fibrils and analyzed the powders by X-ray powder diffractometry. However, we have failed to detect any diffraction patterns arising from new phases other than those of zeolite-A crystals. This indicates that the avidin does not crystallize during fibrillation with biotin-Z. The result also eliminates our previously proposed possibility that the fibrillation takes place by crystallization of β-glucosidase on the surface of D-glucose tethering zeolite-A. However, there is still the possibility that the number of protein layers in the crystalline form is too small to be detected by the conventional X-ray powder diffractometry. In any rate, the mechanism for such intriguing fibrillation still remains open. III. Formation of Discrete Zeolite Clusters at Low A/Z Ratio. While examining the effect of the A/Z ratio on
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Figure 6. SEM images showing the discrete clusters of biotin-Z at the lower (A) and higher magnifications (B) produced at A/Z ratio of 0.1.
the morphology of the fibrils with increasing the ratio from 0.2, we also decreased the ratio to 0.1 to examine the effect of the low A/Z ratio on the morphology of the resulting avidin-biotin-Z composite. Under such a condition of very low A/Z ratio, we found that the zeolite crystals rather aggregate into discrete clusters of zeolite crystals, as shown in Figure 6, instead of undergoing fibrillation. This result indicates that avidin indeed serves as the linker for biotin-Z building blocks since no such clustering occurs in the absence of avidin. This result also emphasizes that there should be enough avidin for fibrillation to occur, and avidin is the one that actually determines the final morphology of the avidin-biotin-Z composite material, either cluster or fibril. Interestingly, there are many small domains, if not all, within the clusters that have three-dimensional (3D) ordering. Obviously, the lack of entire 3D ordering is attributed to the nonuniformity in the size of the zeolite building blocks. We therefore predict that if zeolite crystals with highly uniform sizes are employed, then large zeolite supercrystals with 3D ordering will be produced under the low A/Z condition. Acknowledgment. We thank the Creative Research Initiatives (CRI) program of the Ministry of Science and Technology (MOST) for financial support. K.-K.K and S.H.U. also thank the Advanced Backbone IT Technology Development Project (IMT-2000-133-2) of the Ministry of Information and Communication of Korea. We extend our thanks to Dr. K. S. Yun at LG Electronics Institute of Technology for helpful discussions. LA020118O
