Langmuir 2007, 23, 1615-1618
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Realizing a Two-Dimensional Ordered Array of Ferritin Molecules Directly on a Solid Surface Utilizing Carbonaceous Material Affinity Peptides Takuro Matsui,† Nozomu Matsukawa,† Kenji Iwahori,‡ Ken-Ichi Sano,‡,§ Kiyotaka Shiba,‡,§ and Ichiro Yamashita*,†,‡,⊥ AdVanced Technology Research Laboratories, Matsushita Electric Industrial Co., Ltd. (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, Tokyo 135-8550, Japan, and Materials Science, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan ReceiVed May 10, 2006. In Final Form: December 1, 2006 A two-dimensional hexagonally close-packed (2D-HCP) array of ferritin molecules with a nanoparticle core was fabricated directly on a carbonaceous solid substrate by genetically modifying the outer surface of the ferritin with carbonaceous materials-specific binding peptides. The displayed peptides endowed ferritins with a specific proteinsubstrate interaction and masked the strong nonspecific interaction. The specific interaction was weak enough to allow ferritins to be rearranged as well as an attractive protein-protein interaction that could be adjusted by selecting the buffer conditions. This method not only produced 2D-HCP arrays of ferritin but also 2D-ordered arrays of independent inorganic nanoparticles after protein elimination that can be applied to floating gate memories.
Introduction Apoferritin, a cage-shaped supramolecule, has attracted considerable interest due to its ability to biomineralize various kinds of homogeneous nanoparticles (NPs) within its inner cavity.1-5 Since the size of synthesized NPs is limited by their inner cavity size of 7 nm, they must be 7 nm in diameter. The outer protein shell can be used to automatically deliver ferritins (apoferritin with a core) to specific locations. By exploiting these advantages, we are now making a metal oxide semiconductor field-effect transistor (MOSFET) equipped with floating-gate nanodots that are fabricated utilizing ferritins. The fabricated MOSFET works as a floating-gate memory (FGM).6,7 In an FGM, the floating nanodot gate is the most important component. Since it is densely distributed at more than 1011/cm2, a dispersed nanodot monolayer is required. A two-dimensional hexagonally close-packed (2D-HCP) ordered array of ferritin molecules that theoretically has a density of 8 × 1011/cm2 would be an ideal solution for this problem. The literature describes successful 2D crystallization of native ferritins at an air-water * Corresponding author. Address: Advanced Technology Research Laboratories, Matsushita Electric Industrial Co., Ltd. (Panasonic), 3-4, Hikaridai, Seika-cho, Kyoto 619-0237, Japan. Telephone number: +81-774-98-2516. fax number: +81-774-98-2515. E-mail address: yamashita.ichiro@ jp.panasonic.com. † Matsushita Electric Industrial Co., Ltd. (Panasonic). ‡ Japan Science and Technology Agency. § Japanese Foundation for Cancer Research. ⊥ Nara Institute of Science and Technology. (1) Mann, S. Nature 1993, 365, 499-505. (2) Douglas, T.; Young, M. Nature 1998, 393, 152-155. (3) Okuda, M.; Kobayashi, Y.; Suzuki, K.; Sonoda, K.; Kondoh, T.; Wagawa, A.; Kondo, A.; Yoshimura, H. Nano Lett. 2005, 5, 991-993. (4) Iwahori, K.; Yoshizawa, K.; Muraoka, M.; Yamashita, I. Inorg. Chem. 2005, 44, 6393-6400. (5) Tsukamoto, R.; Iwahori, K.; Muraoka, M.; Yamashita, I. Bull. Chem. Soc. Jpn. 2005, 78, 2075-2081. (6) 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-L3. (7) Hikono, T.; Matsumura, T.; Miura, A.; Uraoka, Y.; Fuyuki, T.; Takeguchi, M.; Yoshii, S.; Yamashita, I. Appl. Phys. Lett. 2006, 88, 023108.
interface.8-10 High-quality ferritin 2D crystals with few defects were fabricated utilizing a divalent cation salt bridge connecting the specific surface positions.8 In some cases, the obtained ferritin 2D crystals were transferred onto a silicon substrate.9,10 However, in this approach, a salt bridge with Cd2+ or Mg2+ ions is required, a critical disadvantage in semiconductor devices. Furthermore, transfer from the air-water interface onto the substrate caused a considerable number of defects and disorder of the 2D crystal. Other disadvantages include patterning difficulty and an inability to control the direction of the crystal axis. Therefore, direct 2D crystal formation of ferritin on the substrate is crucial for floating nanodot gate fabrication. We addressed this issue by genetically modifying the outer surface of ferritin with target-specific affinity peptides.11 The ferritin with the peptides shows a specific interaction with the target inorganic surface.11,12 We therefore expected that the specific protein-substrate interaction would attract ferritin to the substrate by preparing an appropriate substrate surface and that a strong nonspecific protein-substrate interaction would be avoided. We also expected that the optimal protein-protein interaction could be controlled by using the peptides and selecting the buffer conditions. In this study, we used the genetically modified ferritin, N1-LF, which has peptide aptamers against carbonaceous materials (NHBP-1)13 at the N-termini of L-type recombinant horse spleen ferritin (Fer0) (Figure 1a),11 and attempted the direct formation of a 2D-HCP array of N1-LF on a solid substrate. We discuss its possible mechanism. (8) Yoshimura, H.; Scheybani, T.; Baumeister, W.; Nagayama, K. Langmuir 1994, 10, 3290-3295. (9) Furuno, T.; Sasabe, H.; Ulmer, K. M. Thin Solid Films 1989, 180, 2330. (10) Yamashita, I. Thin Solid Films 2001, 393, 12-18. (11) Sano, K.; Ajima, K.; Iwahori, K.; Yudasaka, M.; Iijima, S.; Yamashita, I.; Shiba, K. Small 2005, 1, 826-832. (12) Hayashi, T.; Sano, K.; Shiba, K.; Kumashiro, Y.; Iwahori, K.; Yamashita, I.; Hara, M. Nano Lett. 2006, 6, 515-519. (13) Kase, D.; Kulp, J. L.; Yudasaka, M.; Evans, J. S.; Iijima, S.; Shiba, K. Langmuir 2004, 20, 8939-8940.
10.1021/la061318t CCC: $37.00 © 2007 American Chemical Society Published on Web 01/11/2007
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Figure 1. (a) Schematic drawing of an N1-LF molecule with an amino acid sequence of N-terminus. (b) TEM image of apoN1-LF with uranyl acetate stain.
Letters solution was spread between the paraffin film and the substrate. After an incubation period of 30 min at 20 °C, the substrate was picked out of the solution, and the protein solution remaining on the surface was removed by centrifugation at 1500 G for 10 min in an airtight microtube. The array produced by this procedure was observed using a field-emission high-resolution scanning electron microscope (FE-SEM, JEOL JSM-7400F, JEOL, Tokyo, Japan) at an operating voltage of 5.0 kV. Dynamic light scattering (DLS) was performed with a Zetasizer Nano-ZS (Sysmex, Hyogo, Japan) operating at 632.8 nm. To remove residual dust and air bubbles, all solutions were filtered through 0.1-µm filter units (Millipore Ultra Free-MC, Massachusetts). The solution temperature was maintained by a Peltier device. Correlation functions were measured over delay times ranging from 1 µs to 1 s for a few minutes.
Results and Discussion
Figure 2. (a) Process scheme of the sandwich method. (b) SEM image of a 2D-HCP ordered array constructed from a single layer of Fe oxide NPs with N1-LF on a silicon substrate covered with hydrophilic carbon film.
Experimental Section We synthesized iron and indium oxide NP cores inside the apoN1LF (see Appendix).3 After core formation, N1-LF multimers were carefully removed and a N1-LF solution containing >95% monomeric molecules was prepared. The protocol for making a 2D-HCP array is as follows (Figure 2a). First, carbon film 10 nm thick was vacuum-deposited on a silicon substrate with a 3-nm-thick thermally oxidized silicon layer. The substrate surface was made hydrophilic by the treatment of low pressure air plasma (HDT-400, JEOL DATUM, Tokyo, Japan) just before use. Second, 0.5 mg/mL of N1-LF (Fe) in several kinds of buffers, including 5.0-50 mM PIPES (pH 7.0), 50 mM HEPES (pH 7.0) with 69 or 149 mM NaCl, 50 mM Bis-Tris (pH 7.0), and 80 mM NaCl, was prepared, and a 5-µL drop of the solution was placed on a paraffin film. The substrate was put on this droplet, and the
Two interactions are necessary to make 2D-HCP monolayer arrays of ferritin. One is the weak attractive force between ferritin and the substrate, and the other is the attractive force between ferritins. To realize these two essential interactions, we genetically modified Fer0 with NHBP-1 peptides on the outer surface. NHBP-1 was selected by a phage display method using singlewalled carbon nanohorns treated with 70% nitric acid.13 NHBP-1 interacts specifically with the hydrophilic carbonaceous surface, and the NHBP-1 peptides attract each other due to their hydrophobicity.11,13 Therefore, N1-LF should potentially have the ability to form a 2D-HCP monolayer array on a carbonaceous surface. This expectation was verified when we observed purified N1-LF in a 50 mM Tris-HCl (pH 8.0) solution by transmission electron microscopy (TEM). By chance, we found small 2Dordered arrays on the hydrophilic carbon film-coated TEM grid (Figure 1b). Furthermore, N1-LF in 50 mM PIPES (pH 7.0 and pH 6.0) solutions made larger 2D-ordered arrays on the TEM grid. Prompted by these results, we studied the most suitable conditions for the formation of a 2D-HCP monolayer array of N1-LF with PIPES (pH 7.0) and designed a protein adsorption protocol. A silicon substrate covered with carbon film was prepared, and the carbonaceous surface was made hydrophilic by hydrophilic treatment. This substrate was expected to show a specific interaction with N1-LF. We then designed a new protein adsorption protocol, named the “sandwich method” (Figure 2a). Small amounts of N1-LF solution were placed on a paraffin film, and the prepared substrate was put onto the droplet. This protocol allows a very small amount of N1-LF solution to cover the entire substrate without drying out. This protocol also minimizes N1LF waste. The incubation time was set at 30 min to ensure that equilibrium protein adsorption was realized. After incubation, a small amount of excessive solution was spun away by centrifugation in an airtight tube, and then the substrate was air-dried. A survey of appropriate buffer conditions was first conducted to control the protein-protein interaction. Using the substrate and the protocol described above, N1-LF (Fe) was deposited on the substrates in a variety of buffer solutions at various pH values and concentrations, and their SEM images were observed. We selected the minimum concentration of both N1-LF and PIPES necessary to form 2D-HCP. Our survey showed that a 12.5 mM PIPES solution at pH 7.0 is optimal to form a 2D-HCP ordered monolayer array. A typical SEM image is shown in Figure 2b. Since SEM cannot visualize protein shells, the independent white dots representing the Fe oxide core can be seen in an ordered array. A 2D-ordered array with a typical domain size of 100 nm2 was formed on parts of the sample surface. Using this procedure, we also confirmed that the formation of 2D-HCP N1-LF (Fe)
Letters
Figure 3. SEM images of a Fe oxide NP (a) with Fer0 that does not have NHBP-1 peptides on a silicon substrate covered with the hydrophilic carbon film and (b) with N1-LF on a silicon substrate covered with hydrophobic carbon film.
was reproducible. There were empty spaces in the array. When the concentration of N1-LF was independently changed from 0.5 to 2.0 mg/mL and that of PIPES was changed from 5 to 50 mM, the obtained 2D-HCP showed the same percentage of defects in the array. Therefore, it was suggested that defects in the array reflect apoferritin (N1-LF without core). Buffer selection was critical. For example, other Good buffers such as HEPES and Bis-Tris could not produce 2D-ordered arrays. Replacing the buffer agent with NaCl showed identical results, which indicated that 2D formation is not only caused by electrostatic shielding. These data suggest that PIPES introduces an attractive force among N1-LF to make a 2D-HCP monolayer array. It was proven that N1-LF can make a 2D-ordered array on a hydrophilic carbonaceous surface using a PIPES buffer. We then carried out the same procedures using the Fer0 (Fe) molecule, which does not have NHBP-1, to confirm that NHBP-1 does in fact endow ferritin with the ability to make a 2D-ordered array. A typical SEM image of Fer0 on the substrate is shown in Figure 3a. A closely packed but non-2D-ordered array was observed. Since proteins usually adhere strongly to the solid surface with nonspecific interaction, the result showed that Fer0 adhered to the hydrophilic carbonaceous surface nonspecifically and formed an amorphous 2D array, suggesting that the NHBP-1 peptides screened the strong nonspecific interaction between the Fer0 and carbonaceous surfaces. We investigated the difference between carbonaceous surfaces with/without hydrophilic treatment to confirm that the specific interaction between N1-LF and the hydrophilic carbonaceous surface is necessary. We carried out sandwich protocols and observed N1-LF (Fe) on the substrate by SEM. Without hydrophilic treatment, N1-LF (Fe) adhered randomly, and a 2Dordered array of ferritin did not form on the substrate (Figure 3b). We also found that N1-LF (Fe) adhered very strongly to the hydrophobic surface, since it could not be removed using an intensive pure water rinse process (data not shown). On the other hand, hydrophilic treatment produced a 2D-ordered array that could be detached by a pure water rinse process (data not shown). These results indicate that hydrophilic treatment endowed the substrate and Na-LF with weak protein-substrate interaction that may allow N1-LF molecules to realign themselves, resulting in the formation of the 2D-HCP array. On the other hand, the interaction between N1-LF and the carbonaceous surface without hydrophilic treatment is too strong for N1-LF to rearrange a 2D-HCP array. For 2D-HCP array formation, it is equally important that N1LF have an attractive interaction when on the surface. The hydro-
Langmuir, Vol. 23, No. 4, 2007 1617
Figure 4. Diffusivities Dc as a function of ferritin concentration Cp in 12.5 mM PIPES, pH 7.0 (circles), and 50 mM PIPES, pH 7.0 (squares). N1-LF (Fe): open symbols with solid lines; Fer0 (Fe): closed symbols with dashed lines.
phobic interaction of displayed peptides can produce an attractive force, but this cannot explain the buffer-dependent 2D-HCP formation. Hence, we investigated the protein-protein interaction of Fer0 (Fe) and N1-LF (Fe) in a PIPES buffer by DLS,14-16 which characterizes the interaction of proteins in the solution as well as static light scattering.17,18 Figure 4 shows diffusivity Dc plotted against ferritin concentration Cp. The positive and negative slopes indicate repulsive and attractive protein-protein interactions, respectively. The absolute value of the slope corresponds to the strength of the interactions. The interaction of Fer0 (Fe) does not vary, but the interaction of N1-LF (Fe) does change significantly from repulsion to attraction when the buffer concentration increases. This attractive force caused by the increased PIPES buffer concentration may explain the additional attractive force between N1-LF for the 2D-HCP array formation. That is, the increase in buffer concentration accompanying the drying process induces an attractive force that makes N1-LF self-assemble into the 2D-HCP array. The role of 50 mM PIPES in the creation of this attractive force is not clear. The symmetry of the PIPES structure may contribute; that is, the sulfonic groups at both ends of PIPES may bridge the N1-LFs. We further examined whether the NP core inside the protein shell affected the 2D-ordered array formation. We synthesized an In oxide NP core in apoN1-LF, and the sandwich protocol was carried out (Figure 5a). A 2D-HCP ordered monolayer array with a typical domain size of 200 nm2 can be visualized on parts of the sample surface. This SEM image showed that different cores do not affect 2D-ordered array formation and that N1-LF with a different material NP core can be made into 2D-ordered arrays. On the basis of this result, we studied whether the absolute PIPES concentration (12.5 mM) or the stoichiometry of PIPES and N1-LF is essential. A diluted solution of optimum condition, namely, 0.07 mg/mL of N1-LF in 1.75 mM PIPES (pH 7.0), was used in the sandwich protocol. SEM observation showed a large 2D-HCP ordered monolayer array (Figure 5b). This result indicated that the stoichiometry of PIPES and N1-LF is essential and that a proper amount of PIPES molecules per one N1-LF (14) Muschol, M.; Rosenberger, F. J. Chem. Phys. 1995, 103, 10424-10432. (15) Muschol, M.; Rosenberger, F. J. Cryst. Growth 1996, 167, 738-747. (16) Rosenberger, F.; Vekilov, P. G.; Muschol, M.; Thomas, B. R. J. Cryst. Growth 1996, 168, 1-27. (17) Abraham, G.; Wilson, W. W. Acta Crystallogr. 1994, D50, 361-365. (18) Petsev, D. N.; Thomas, B. R.; Yau, S. T.; Vekilov, P. G. Biophys. J. 2000, 78, 2060-2069.
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Letters
array with N1-LF involves the prevention of aggregation by means of the electrostatic repulsion force in the solution, a weak and specific attractive interaction between the peptide-substrate surface, and the protein-protein attractive force that develops during the drying process under PIPES buffer. A FGM is now under development that employs nanodot arrays fabricated using the method described here. Acknowledgment. We thank Mitsuhiro Okuda, Kazuaki Nishio, Taeko Ishikawa, and Yoko Suzumoto at ATRL Matsushita Electric Industrial. Co., Ltd. for the formation of NP cores in N1-LF. Yoshikazu Kumashiro and Masahiko Hara at the Tokyo Institute of Technology kindly advised us on measurement of the DLS data. We thank Daisuke Kase at JFCR for fruitful discussions. This work was supported by the Leading Project of the Ministry of Education, Culture, Sports, Science and Technology, Japan.
Appendix
Figure 5. SEM image of a 2D-ordered array constructed from a single layer of In oxide NPs with N1-LF on a silicon substrate covered with hydrophilic carbon film (a) using optimum solution conditions: 0.5 mg/mL of N1-LF in 12.5 mM PIPES (pH 7.0); and (b) using a seventh dilution of the optimum solution conditions: 0.07 mg/mL of N1-LF in 1.75 mM PIPES (pH 7.0)
increased the attractive protein-protein interaction in the drying process to form a 2D-HCP array. In summary, we successfully created a single-layer 2D-HCP array of ferritin with Fe or In oxide NP cores by genetically modifying its outer surface with NHBP-1s. A 2D-HCP array of N1-LF was made directly on the thermally oxidized silicon substrate covered with a hydrophilically treated thin carbon layer. We showed that NHBP-1 changes these interactions regardless of core material. This process of directly forming a 2D-ordered
We overproduced and purified recombinant apoN1-LFs (without NP cores) and synthesized metal oxide cores inside the cavity.3 Iron oxide NP cores were formed in the apoN1-LF cavities by incubating 0.5 mg/mL of apoN1-LF in 80 mM HEPES (pH 7.5) and 5 mM ammonium iron sulfate at room temperature for 2 h. After core formation, N1-LF (Fe) molecules were purified by gel filtration (HiPrep 26/60 Sephacryl S-300 HR, GE Healthcare Bio-Sciences). N1-LF multimers were carefully removed, and N1-LF solution (∼5.0 mg/mL of protein in 2 mM Tris-HCl buffer, pH 8.0) containing >95% monomeric molecules and
