Langmuir 2009, 25, 3327-3330
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Hexagonal Close-Packed Array Formed by Selective Adsorption onto Hexagonal Patterns N. Matsukawa,† K. Nishio,† K. Sano,‡,§ K. Shiba,‡,§ and I. Yamashita*,†,‡,| AdVanced Technology Research Laboratories, Panasonic, 3-4 Hikaridai, Seika-cho, Soraku-gun, Kyoto 619-0237, Japan, CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan, Department of Protein Engineering, Cancer Institute, Japanese Foundation for Cancer Research, 3-10-6 Ariake, Koto-ku, Tokyo 135-8550, Japan, and Materials Science, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan ReceiVed September 29, 2008. ReVised Manuscript ReceiVed January 29, 2009 A patterned two-dimensional hexagonally ordered array of ferritin molecules, the outer surfaces of which had been genetically modified by titanium (Ti) specific binding peptides (minT1-LF), was realized in a self-assembling manner on a hexagonal Ti thin film island made on a silicon substrate. The optimum degree of order was realized at the pH with the maximum selectivity of minT1-LF adsorption on the Ti surface with respect to the silicon dioxide (SiO2) surface. Quartz crystal microbalance (QCM) measurement revealed that minT1-LF adsorbed onto the Ti surface strongly and irreversibly, but adsorbed onto the silicon dioxide surface weakly and reversibly. It was suggested that the concentration of minT1-LF on the Ti pattern promotes hexagonal close-packed ordering and axis aligning.
Introduction Increasingly, intensive research is being conducted on the self-assembly of organic and biomolecular systems to fabricate nanostructures smaller than those possible via conventional lithography techniques. Numerous molecules have the potential to act as nanoconstruction elements for self-assembly into nanostructures. Proteins are among the most promising elements. Their monodispersed size and identical structure at atomic resolution made them ideal nanobuilding-block candidates and it has already been shown that they self-assemble into higherorder structures with sophisticated functions in nature. Furthermore, their characteristics and functions can be modified by genetic engineering. Previous studies have reported the successful construction of nanometric functional structures created thorough the use of proteins.1-4 In the nanoelectronic devices field, we have pioneered a technique using ferritins to fabricate nanodot arrays for floating nanodot gate memory.5-8 Ferritin is a cageshaped protein with the ability to biomineralize various materials in its cavity, which has a diameter of roughly 7 nm.9-16 A high* Corresponding author. Address: Advanced Technology Research Laboratories, Panasonic, 3-4 Hikaridai, Seika-cho, Soraku-gun, Kyoto 6190237, Japan. Telephone number: +81-774-98-2516. Fax number: +81-77498-2515. E-mail address:
[email protected]. † Panasonic. ‡ Japan Science and Technology Agency. § Japanese Foundation for Cancer Research. | Nara Institute of Science and Technology.
(1) Lee, S. W.; Mao, C.; Flynn, C. E.; Belcher, A. M. Science 2002, 296, 892. (2) Banerjee, I. A.; Yu, L.; Matsui, H. J. Am. Chem. Soc. 2005, 127, 16002. (3) Pum, D.; Neubauer, A.; Gyorvary, E.; Sara, M.; Sleytr, U. B. Nanotechnology 2000, 11, 100. (4) Sugimoto, K.; Kanamaru, S.; Iwasaki, K.; Arisaka, F.; Yamashita, I. Angew. Chem. 2006, 45, 2725. (5) Yamashita, I. Thin Solid Films 2001, 393, 12. (6) Hikono, T.; Matsumura, T.; Miura, A.; Uraoka, Y.; Fuyuki, T.; Takeguchi, M.; Yoshii, S.; Yamashita, I. Appl. Phys. Lett. 2006, 88, 023108. (7) Miura, A.; Hikono, T.; Matsumura, T.; Yano, H.; Hatayama, T.; Uraoka, Y.; Fuyuki, T.; Yoshii, S.; Yamashita, I. Jpn. J. Appl. Phys. 2006, 45, L1. (8) Miura, A.; Tsukamoto, R.; Yoshii, S.; Yamashita, I.; Uraoka, Y.; Fuyuki, T. Nanotechnology 2008, 19(25), 255201. (9) Loewus, M. W.; Fineberg, R. A. Biochim. Biophys. Acta 1957, 26, 441. (10) Harrison, P. M.; Fischbach, F. A.; Hoy, T. G.; Haggis, G. H. Nature 1967, 216, 1188. (11) Douglas, T.; Stark, V. T. Inorg. Chem. 2000, 39, 1828.
density monolayer of ferritin was formed in a self-assembling manner on the SiO2 surface of a Si substrate, and protein elimination left independent biomineralized nanodots that were used as the floating gate memory’s charge storage nodes. For future bottom-up nanodevice constructions, more sophisticated controls of two-dimensional (2D) arrays are necessary. So far, we have reported high-density ferritin adsorption on patterned areas by utilizing electrostatic interaction,17-19 and a hexagonal close-packed (HCP) array formation by way of N-termini modification with artificial carbonaceous-material affinity peptides.20,21 However, an HCP array on a patterned area with controlled axis of the array was not achieved. In this study, we employed recombinant ferritin-displaying hexapeptides that have a specific affinity to titanium (Ti)22 to address this subject and demonstrated the formation of a 2D hexagonally ordered array of ferritin molecules on a hexagonal Ti thin film island.
Experimental Section Ti-binding ferritins (minT1-LF) were overproduced and purified. Ferrihydrite cores or cobalt oxide cores were artificially synthesized in minT1-LF. The details are described elsewhere.15,19,23,24 (12) Tsukamoto, R.; Iwahori, K.; Muraoka, M.; Yamashita, I. Bull. Chem. Soc. Jpn. 2005, 78, 2075. (13) Okuda, M.; Iwahori, K.; Yamashita, I.; Yoshimura, H. Biotechnol. Bioeng. 2003, 84, 187. (14) Yamashita, I.; Hayashi, J.; Hara, M. Chem. Lett. 2004, 33, 1158. (15) Iwahori, K.; Yoshizawa, K.; Muraoka, M.; Yamashita, I. Inorg. Chem. 2005, 44, 6393. (16) Wong, K. K. W.; Mann, S. AdV. Mater. 1996, 8, 928. (17) Yoshii, S.; Yamada, K.; Matsukawa, N.; Yamashita, I. Jpn. J. Appl. Phys. 2005, 44, 1518. (18) Kumagai, S.; Yoshii, S.; Yamada, K.; Matsukawa, N.; Fujiwara, I.; Iwahori, K.; Yamashita, I. Appl. Phys. Lett. 2006, 88, 153103. (19) Yamashita, I.; Kirimura, H.; Okuda, M.; Nishio, K.; Sano, K. I.; Shiba, K.; Hayashi, T.; Hara, M.; Mishima, Y. Small 2006, 2, 1148. (20) Matsui, T.; Matsukawa, N.; Iwahori, K.; Sano, K. I.; Shiba, K.; Yamashita, I. Langmuir 2007, 23, 1615. (21) Kase, D.; Kulp, J. L.; Yudasaka, M.; Evans, J. S.; Iijima, S.; Shiba, K. Langmuir 2004, 20, 8939. (22) Sano, K. I.; Sasaki, H.; Shiba, K. Langmuir 2005, 21, 3090. (23) Takeda, S.; Ohta, M.; Ebina, S.; Nagayama, K. Biochim. Biophys. Acta 1993, 1174, 218. (24) Sano, K. I.; Ajima, K.; Iwahori, K.; Yudasaka, M.; Iijima, S.; Yamashita, I.; Shiba, K. Small 2005, 1, 826. (25) Sano, K. I.; Shiba, K. J. Am. Chem. Soc. 2003, 125, 14234.
10.1021/la8032012 CCC: $40.75 2009 American Chemical Society Published on Web 02/19/2009
3328 Langmuir, Vol. 25, No. 6, 2009 We used two kinds of substrates. One was a thermally oxidized silicon substrate, half of which was covered by Ti thin film. The other one was a thermally oxidized silicon substrate with hexagonal Ti patterns. We fabricated hexagonal holes measuring 65 nm per side in electron beam (EB) resist layers (ZEP520, Zeon; positive type) on a thermally oxidized Si substrate using EB lithography (ELS-7500, Elionix). EB-exposed samples were developed in n-amyl acetate. Ti thin films were deposited onto resist-patterned substrate by RF magnetron sputtering from a pure Ti metal target (99.98%, Kojundo Chemical Laboratory), and its thickness was controlled at 1 nm. The EB resist was removed in n-dimethylacetamide and washed in an ultrasonic cleaner successively in pure water, ethanol and acetone. Finally, the Ti-patterned substrate was cleaned with UV/ ozone cleaner just before the adsorption process. Patterned islands were examined using an AFM in tapping mode (SPI4000, SII). We prepared minT1-LF solutions with concentrations of 0.1, 0.5, 2.0 mg/mL in a mixture solution of 2-(N-morpholino)ethanesulfonic acid (MES), (hydroxymethyl)aminomethane (Tris), and Tween-20 (1.0 vol %, ICI) at pH 6.7, 7.4, 7.8, 8.0, and 8.2. Tween-20 was always added at 1.0 vol % on the basis of our preliminary experiments. The concentration of the buffer was controlled so that ionic strength would be 50 mM in consideration of the pKa of MES and Tris. A solution containing minT1-LF was placed on the substrate and left for 1 h at room temperature. The substrate was washed with buffer solution three times and pure water two times. After washing, the substrate was placed in an airtight tube and centrifuged at 2000 × g for 30 s to remove excess water. The samples were observed by field-emission scanning electron microscopy (FE-SEM, JSM-7400F, JEOL), and metal oxide cores in the adsorbed ferritins were visualized. The number of ferritin cores in an area of 200 × 200 nm2 was counted. If there were fewer than 100, the numbers from three areas were averaged. Fast Fourier transform (FFT) was carried out using ImageJ software (NIH) from the 150 × 150 nm2 areas cut from SEM images that included the whole Ti pattern. A circular signal distribution of an FFT image in the radius of the spots was processed to obtain the average and standard deviation of the signal. Signals less than the sum of the average and standard deviation were deleted as background noise. The radial signal distribution was calculated, and the correlated length in the SEM image was estimated. The correlated length is expected to be the minimum distance between the dots in the case of a randomly packed domain and is expected to be the lattice constant in the case of an HCP array (see Supporting Information). The adsorption behavior of minT1-LF onto the SiO2 and Ti surfaces was monitored using a quartz crystal microbalance (QCM-D300, Q-Sense) with a SiO2-coated sensor and Ti-coated sensor.
Results and Discussion The adsorption difference of minT1-LF onto Ti and SiO2 surfaces at various solution pHs (from 6.7 to 8.2) and protein concentration was first investigated using the thermally oxidized silicon substrate half-covered with Ti. Our previous work suggested that the interaction between minT1-LF and the Ti surface is due to electrostatic attraction,25,26 indicating that pH must influence the attractive force between the minT1-LF and Ti surface and, in turn, selective adsorption. The SEM images clearly showed the minT1-LF adsorption behavior onto the Ti and SiO2 surface, and subsequently the numbers of minT1-LF adsorbed in the 200 × 200 nm2 area were counted. The results are summarized in Figure 1. At pH 6.7-7.4, the counting was marginal because of the formation of occasional multilayers or aggregates, but we were able to reasonably estimate the number. The minT1-LFs on Ti were distributed evenly, and the numbers remained fairly constant at around 200, which is 60-70% of the theoretical maximum when they were hexagonally closed-packed. On the SiO2 surface, minT1-LFs were rarely observed except at (26) Hayashi, T.; Sano, K. I.; Shiba, K.; Kumashiro, Y.; Iwahori, K.; Yamashita, I.; Hara, M. Nano Lett. 2006, 6, 515.
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Figure 1. Counts of observed cores (left axis) on Ti (open circles) and on SiO2 (squares) in an area of 200 × 200 nm2, and count ratios of cores on Ti over on SiO2 (closed circle, right axis) using a solution of 2.0 mg/mL minT1-LF in 50 mM MES-Tris buffer. At pH 6.7 and 7.4, an aggregation or multilayer of minT1-LF was occasionally observed.
pH 8.2. The adsorption number below pH 8.0 was less than 1 and, at pH 7.8 the number was as low as 0.17. At pH 8.2, the number increased sharply to as high as 18. The adsorption selectivity, defined by the ratio of counts of Ti over the SiO2 surface, showed a single peak at pH 7.8. Judging from these results, a solution of pH 7.8 is optimal for selective adsorption. The effect of the concentration of minT1-LF on selectivity was also investigated. The same adsorption processes onto Ti and SiO2 were carried out at a concentration range of 0.1 to 2.0 mg/mL at pH 7.8 and 8.0, and the adsorbed minT1-LFs were counted. The numbers showed little difference, and the selectivity was unchanged, except for 0.1 mg/mL at pH 7.8 where the number adsorbed on Ti is a little smaller. The adsorption flux density of minT1-LF is assumed to be smallest at pH 7.8 and 0.1 mg/mL as indicated by the lowest protein concentration, and the smaller diffusion constant at pH 7.8 than pH 8.0 is due to a greater repulsive force among minT1-LFs. Therefore, one possible cause of the exception was that adsorption had not reached the saturated state. But QCM measurement of the adsorption behavior of 0.1 mg/mL at pH 7.8 onto the Ti-coated sensor showed that the adsorption was saturated within 20 min. The difference in adsorption behavior at pH 7.8 and pH 8.0 may suggest that electrostatic interaction between minT1-LF and Tween-20 was among the possible causes. However, the cause of the exception remains unclear. We produced hexagonal nanometer sized island-patterns, expecting that minT1-LFs would form HCP arrays on them, if they cover the pattern fully. HCP has 6-fold symmetry, and the axis of the HCP array would be aligned to each side of the hexagon. The length of each side of the hexagon was designed to be around 5 times longer than that of minT1-LF. Atomic force microscopy (AFM) measurements showed the clear hexagons of the Ti pattern with a smooth surface. The thickness was around 1.5 nm, which was thicker than the deposited metal Ti thickness (1 nm) because of oxidation. The diagonal length of hexagonal pattern was around 130 nm, which is in good agreement with the designed length. Figure 2a shows a typical SEM image of a Ti hexagonal pattern with a monolayer of minT1-LF, which was formed with 2.0 mg/mL minT1-LF in 50 mM MES-Tris at pH 8.0 with 1 vol % Tween-20. A hexagonally ordered array of ferritin cores was observed visually. Even though the protein shells could not be observed, this SEM image indicates that an HCP array of ferritin
Letters
Figure 2. (a) SEM image of minT1-LF with Fe oxide cores adsorbed on the hexagonal Ti pattern using a solution of 2.0 mg/mL minT1-LF in 50 mM MES-Tris buffer at pH 8.0 with 1 vol % Tween-20. (b) 2D FFT result of the SEM image shown in panel a. (c) 2D FFT result after subtracting background, taking into account the average and standard deviation of the signal around the spots. The lines of 4-fold symmetry around the center spot are artifacts of the XY scan of SEM.
Figure 3. 2D FFT results from SEM images of minT1-LF with Fe oxide selectively adsorbed onto the hexagonal Ti pattern. From left to right, pH is 6.7, 7.4, 7.8, 8.0, and 8.2 respectively. From top to bottom, concentration of minT1-LF is 2.0, 0.5, and 0.1 mg/mL.
molecules had been formed, and the axis of array correlated with the direction of the Ti pattern. The fast Fourier analysis (FFA) image of Figure 2a is shown in Figure 2b. Six spots can be seen clearly, corresponding to the first order of the HCP array. To extract the peak positions of the spots, signals less than the sum of the average and standard deviation were designated as background noise and deleted (Figure 2c, see Supporting Information). The calculated periodic distance was estimated to be about 11 nm, which was nearly the lattice constant of the HCP array, 3/2 of the diameter of a ferritin molecule. Comparisons among the FFT results from 2.0, 0.5, and 0.1 mg/mL minT1-LF, and pH 6.7, 7.4, 7.8, 8.0, and 8.2 are shown in Figure 3. Concerning the pH effect on the degree of order, the sharpest spots were observed at pH 7.8 and 8.0. As for the concentration of minT1-LF, there was little difference between 2.0 mg/mL and 0.5 mg/mL at pH7.8 and 8.0, whereas spots at 0.1 mg/mL were inhomogeneous. The sharpest and clearest spots were obtained at pH 7.8 and a concentration of 0.5 mg/mL. The results showed that the optimum degree of order was realized at the pH where maximum adsorption selectivity was achieved. To investigate the effect of Ti hexagonal patterns on HCP array formation, adsorption of minT1-LFs onto a flat Ti thin film was carried out at pH 7.8 at a concentration of 0.5 mg/mL. Figure 4 shows the SEM images of ferritins with Fe oxide core adsorbed onto flat Ti thin film (Figure 4a) and Ti hexagon islands surrounded by SiO2 (Figure 4b). On the flat Ti surface there were ordered domains that were much smaller than the Ti hexagonal pattern with random axes (Figure 4a). FFT analysis of Figure 4a in the area of 150 × 150 nm2 exhibits an almost homogeneous halo pattern at the radius of 1/12 nm-1, and the correlated length is estimated to be 12 nm, which is identical to the minT1-LF diameter. It was indicated that the domains were too small or had
Langmuir, Vol. 25, No. 6, 2009 3329
Figure 4. SEM images of minT1-LF with Fe oxide cores after adsorption onto a flat Ti thin film and thermally oxidized silicon substrates with Ti hexagonal patterns using a solution of 0.5 mg/mL minT1-LF in 50 mM MES-Tris buffer at pH 7.8 with 1 vol % Tween-20. (a) Substrate with flat Ti thin film. The hexagon is the same size as the Ti pattern in panel b. (b) Thermally oxidized Si substrate with hexagonal Ti patterns.
inferior spatial regularity. On the other hand, on a hexagonal Ti pattern, a hexagonally ordered array with a controlled axis was formed (Figure 4b). It is also confirmed by FFT analysis that the correlated length was 12% shorter than miniT1-LF (12 nm), which is nearly equal to the lattice constant of the HCP array of minT1-LF. The results indicated that the hexagonal Ti pattern created the HCP array and aligned its axis. The effects of a large Ti pattern’s long edge on SiO2 were investigated, but no difference in dot density or in the degree of order of dots was observed between near the edge and the inner region (see Supporting Information III). The role of the surfactant, Tween-20, in the selective adsorption was also studied. QCM with dissipation monitoring (QCM-D) measurements of the adsorption behavior of minT1-LF onto the Ti-coated sensor and SiO2-coated sensor were performed. In the previous study, it was shown that minT1-LF has a strong affinity to negatively and positively charged groups of the Ti substrate and that minT1-LF has moderate affinity to the negatively charged group of SiO2.25,26 Under our experimental conditions, minT1LFs adsorbed almost equally onto the SiO2 and Ti surfaces. It was also shown that rinsing washed out minT1-LFs on the SiO2, while minT1-LFs on the Ti could not be washed out. The dissipation data showed that Tween-20 adsorbs both onto SiO2 and Ti, but more so onto SiO2. The results indicated that TBP-1 penetrated the thin Tween-20 layer and bound the SiO2 and Ti surfaces. The interactions were weakened by Tween-20. As a result, minT1-LFs on the SiO2 surface adsorbed reversibly and were easily washed away. The strong specific interaction kept minT1-LFs remaining on the Ti surface. Thus, it was concluded that Tween-20 increased the selectivity. On the basis of the above results, it is indicated that highly selective adsorption onto a nanometric confined area surrounded by reversibly adsorbed minT1-LFs on SiO2 concentrates the minT1-LF on the pattern, resulting in the growth of an HCP array. The existence of the small domains and gaps in Figure 4a show that there are short-range attractive forces and long-range repulsive forces between minT1-LFs. Those results suggest that one of the possible mechanisms of HCP array formation is as follows. In Figure 4a, the distribution of domains and gaps was determined by the total energy and the nuclei of minT1-LF initially adsorbed. Because the minT1-LFs were reversibly adsorbed onto SiO2 surrounding the Ti pattern, the domain on SiO2 could be rearranged to shift to a more stable position against the domain on the Ti pattern with regard to total energy. This rearrangement and the highly selective adsorption onto Ti concentrate minT1LFs onto the Ti pattern. The result that the axis of the HCP array was controlled by the shape of the Ti pattern suggests that the corner edge of the hexagonal pattern initiates the nucleation because the line edge shows little effect on HCP formation.
3330 Langmuir, Vol. 25, No. 6, 2009
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Conclusion
on the Ti pattern results in a hexagonally ordered array. This method of forming an ordered and oriented array at a designated position in a self-assembling manner is very easy and represents the first step in future bottom-up construction of self-organized nanodevices.
The recombinant ferritin, minT1-LF, showed an affinity to the Ti surface and could selectively adsorb onto Ti patterns along with the blockage of nonspecific adsorption with a nonionic detergent. The selective adsorption onto a hexagon Ti thin film island on SiO2 realized a patterned 2D HCP array with the controlled axis in a self-assembling manner. The optimum degree of order was realized when high selectivity of minT1-LF adsorption on Ti to SiO2 was achieved. The need for a hexagonal Ti pattern for large HCP array formation with a controlled axis suggests nucleation by the corner edge of the hexagonal pattern because the long line edge of the large Ti pattern showed little effect on HCP formation. QCM measurement showed that minT1LF was irreversibly adsorbed onto the Ti island, while minT1LF was reversibly adsorbed onto the SiO2 surface under the same conditions. It is suggested that concentration of minT1-LF
Acknowledgment. We thank Mr. N. Okamoto at NAIST for the overproduction, purification, and core-formation of Ti-binding ferritins. This work was supported by the Leading Project of Ministry of Education, Culture, Sports, Science and TechnologyJapan. Supporting Information Available: Details of the fabrication of hexagonal Ti patterns, signal processing of FFT images, and analyzing the long line edge of Ti/SiO2. This material is available free of charge via the Internet at http://pubs.acs.org. LA8032012
