pubs.acs.org/NanoLett
Fabrication of Aligned Magnetic Nanoparticles Using Tobamoviruses Mime Kobayashi,†,‡ Munetoshi Seki,§ Hitoshi Tabata,§ Yuichiro Watanabe,| and Ichiro Yamashita*,†,‡,⊥ †
Graduate School of Materials Science, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan, ‡ CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama, 332-0012, Japan, § Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan, | Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan, and ⊥ Advanced Technology Research Laboratories, Panasonic Co., Ltd., 3-4 Hikaridai, Seika, Kyoto 619-0237, Japan ABSTRACT We used genetically modified tube-shaped tobamoviruses to produce 3 nm aligned magnetic nanoparticles. Amino acid residues facing the central channel of the virus were modified to increase the number of nucleation sites. Energy dispersive X-ray spectroscopy and superconducting quantum interference device analysis suggest that the particles consisted of Co-Pt alloy. The use of tobamovirus mutants is a promising approach to making a variety of components that can be applied to fabricate nanometerscaled electronic devices. KEYWORDS TMV, nanowires, nanoparticles, magnet
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or the fabrication of nanometer-scaled devices, homogeneous particles and wires with diameters less than 10 nm are necessary as nanosized building blocks. In addition to fine control of the size and shape, it is also required that nanoparticles and nanowires can be selectively placed onto predetermined positions with designated directions, such as bridging electrodes made on substrates, which is a key bottom-up fabrication technique, a directed self-assembly. Since biological supramolecules have identical dimensions and their surfaces can be genetically or chemically modified to change their adhesive properties, the use of tube-shaped biotemplates offers a solution which meets the above-mentioned requirements.1,2 Nanoparticles made using biotemplated methods have already been successfully utilized to fabricate memory devices.3-6 Tobamovirus is a plant virus that is among the favored biotemplate proteins for making nanoparticles and nanowires. Tobacco mosaic virus (TMV) and tomato mosaic virus (ToMV) are components of the tobamovirus species.7 Tobamovirus particles are 300 nm in length with outer and inner diameters of 18 and 4 nm, respectively (Figure 1).8,9 Each particle is composed of a genomic single-stranded RNA and 2130 identical coat proteins. Forty-nine coat proteins make three helical turns around the RNA, and about 44 of these units constitute the virus, about 132 turns with 2130 () 49 × 44 - 26) coat proteins. Each unit is 6.9 nm in length,
resulting in a 300 nm overall length of the virus. Genomic RNA consists of 6390 bases, and one coat protein makes contact via three bases of the RNA molecule. RNA molecules make a helical tube of about 5 nm in diameter, and they are not directly juxtaposed toward the central channel of the virus. Chemical and genetic modifications have been attempted in order to add functional properties to the outer surface of TMV.10-14 Metal depositions to the outer surface of TMV have also been achieved, and their electrical characteristics and potential use as battery electrodes have been reported.15-18 However, in general, it is difficult to control the size of nanoparticles or nanowires when they are formed outside of TMV. In addition, various new functionalities cannot be added when metals cover the outer surface of TMV. As for the mechanisms of biomineralization on the surface of a protein, it has been shown that the electrostatic interactions between source ions and the protein surface are important. In the case of cage-shaped proteins, it has been proposed that highly condensed source ions create nuclei and the inner surface of the protein acts as a self-catalyst.2 The importance of a nucleation site has also been demonstrated by exposing clusters of four negatively charged glutamic acid residues as nucleation sites on the outer surface of bacteriophage M13, which resulted in the formation of Co3O4 nanoparticles.19 Utilizing the central channel of TMV, the formation of Ag nanoparticles has been reported.20 In related studies, the formation of metallic nanowires such as Cu, Ni, Co, Co/Pt, and Fe/Pt has also been reported.13,21,22 The inner surface of tobamoviruses consists of helically stacked coat proteins,
* Correspondence should be addressed to: Ichiro Yamashita, Graduate School of Materials Science, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan; e-mail,
[email protected]; phone, +81-743-726135; fax, +81-743-72-6196. Received for review: 07/26/2009 Published on Web: 02/16/2010 © 2010 American Chemical Society
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DOI: 10.1021/nl902405s | Nano Lett. 2010, 10, 773–776
FIGURE 1. Structure of a tobamovirus. (a) Schematic illustration of tobamovirus. (b) Top view of the virus. (c) Atomic coordination of a coat protein subunit of the virus (PDB:2OM3) prepared with PyMol.24 One helical turn consists of 161/3 coat proteins. In (c), the four residues that were mutated in this study are indicated by red circles. The RNA molecule is also included in the model. (d) Sequence comparison between coat proteins of TMV (U1) and tomato mosaic virus (ToMV). Amino acid residues different between two species are highlighted. Amino acid residues which are facing inner or outer virus surfaces are indicated by boxes. Arrowheads indicate the four residues that were mutated in this study.
and only a limited number of amino acid residues face the central channel. Introducing a single amino acid change in coat proteins would significantly affect the environment of the central channel, which in turn leads to the formation of nanoparticles with new characteristics.20 In this paper, tomato mosaic virus (ToMV) was genetically modified to introduce point mutations in the region facing the inner surface of ToMV so that the number of positive (lysine: K) or negative (asparatic acid: D) charges are increased on the inside in order to generate additional nucleation sites. It turned out that increasing the positive charge at a specific site offers more sites for nucleation, resulting in the growth of aligned nanoparticles inside the virus. Genetically modified ToMVs are useful not only as templates for making nanomaterials but also for analyzing the mechanisms of biomineralization on the protein surface. In previous studies regarding metal deposition using tobamoviruses, the U1 (vulgare) species of tobacco mosaic virus has been used almost exclusively. Because ToMV can be considered a “natural mutant” of U1, we first tried Pt/Co nanoparticle deposition using ToMV. U1 and ToMV have the same overall tube structure, but different amino acid residues are exposed to their inner surfaces (Figure 1). As a result of reducing (NH4)2Co(SO4)2 and K2PtCl4 with NaBH4 in the presence of ToMV, nanowire was formed inside the central channel (Figure S1 in Supporting Information) (details in the experimental methods section in Supporting Information). These results were similar to those obtained for U1,22 © 2010 American Chemical Society
although the average length of nanowire was longer when using ToMV. Because higher ToMV nucleation properties were implied, we used the virus for further modifications. We attempted to make recombinant ToMV virus-like particles with inner-surface residues modified according to the structural information of tobamovirus (Figure 1).9 The model illustrates that side chains of serine at position 101 (S101; asparagin in U1) and threonine at 103 (T103) are juxtaposed toward the central channel. These residues were changed to lysine (Lys), under the assumption that an increase in positive charge would result in more sites for nucleation and enhance the incorporation of negatively charged PtCl42- ions into the channel. In addition to S101 and T103 residues, two glutamic acid residues in the vicinity of the channel at position 97 and 106 (E97 and E106) were changed to lysine residue, respectively. Genomic RNA, which is integral to the helical shape of the virus, is positioned away from these residues (Figure 1). It was expected that these mutations would not interfere with the overall virus structure. Mutant virus-like particles were obtained in all cases except for T103K mutant (Figure 2). Threonine residue is conserved between U1 and ToMV, and it may be important not to have positively charged residue at this position to maintain the overall structure (Figure 1). Changing negatively charged glutamic acid residues to lysine was permissive probably because those residues are not directly facing the inner surface. The length of the virus-like particles was apparently not affected, resulting in 300 nm long particles (Figure 2a). 774
DOI: 10.1021/nl902405s | Nano Lett. 2010, 10, 773-–776
FIGURE 3. TEM images of aligned nanoparticles formed inside S101K (a, b) and E106K (c, d) mutants. TEM images stained with Au-glucose (a, c) or PTA (b, d) are shown.
FIGURE 2. Inner-surface modified ToMV mutants. (a) TEM images of purified wild-type (wt) and mutant virus-like structures stained with 3% phosphotungstic acid (PTA). Scale bar is 100 nm. (b) A list of the mutants constructed.
To investigate whether increased negative charges inside ToMV would affect metal deposition, S101 and T103 residues were next changed to aspartic acid (Asp). However, no virus-like particle could be purified from tobacco plants infected with those mutant RNAs. This result suggests that adding negative charges to an already negatively charged inner-surface is fatal to the virus. This is probably because the additional negative charges disrupt formation of the helical structure in TMV assembly. The mutants that were constructed are summarized in Figure 2b. When Pt/Co deposition was attempted using lysine mutants, S101K and E106K formed Pt/Co nanoparticles inside the channel instead of nanowires (Figure 3). The samples were first stained with Au-glucose, which does not stain the inside of the channel (Figure 3, panels a and c). Because formation of Pt or Au particles on the outer surface of TMV has been reported,4,14,18,20 we stained the samples with phosphotungstic acid (PTA), which allows clearer visualization of proteins (Figure 3 panels b and d). The PTA-stained transmission electron microscopy (TEM) images indicate that nanoparticles are formed inside the channel (Figure 3, panels b and d). The results support our assumption that an increase in positive charge results in more sites for nucleation and enhances the incorporation of negatively charged PtCl42- ions into the channel. The aligned nanoparticles spanned a total length of up to 250 nm with an average length of 75 nm, similar to the length of the nanowires formed inside wild-type (wt) ToMV. No metal deposition was observed inside E97K mutant. The mutation may have affected the structure in such a way that lysine residues are not available as nucleation sites. The © 2010 American Chemical Society
FIGURE 4. High-resolution TEM image and EDS spectrum of aligned nanoparticles formed inside E106K mutant: (a) TEM image without staining; (b) EDS spectrum of nonstained nanoparticles.
structural change resulted in a slight bend of the mutant virus-like particles as evident in Figure 2a. The nanoparticles inside E106K were not aligned in a perfectly straight line, and the high-resolution TEM image showed the diameter of each particle to be about 3 nm. These results suggest that the diameter of the channel had been increased as a result of nanoparticle formation (Figure 4). The distorted form may have been caused by spontaneous nucleation at many positions, which in turn caused the particles to grow and enlarge the inner channel, resulting in bent virus-like structures. Even under such stress, the presence of genomic RNA may have played a role in maintaining the tube-shaped structure of ToMV. When Co2+ solution was omitted from the reaction, smaller particles were formed inside E106K (Figure S2a in Supporting Information). Because ordinary nanowires were formed inside wt ToMV under these conditions (Figure S2b in Supporting Information), it is likely that the mutation increased the number of nucleation sites for Pt to form. Pt particles appear to be lined in two rows. This suggests that the nucleation occurs at some 775
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Because they allow control of nanoparticle size and fabrication of nanoparticle arrays using self-assembly, these components can be applied to devices such as high sensitivity magnetic field sensors and high-density, low-power magnetic logic gates.23 Verification of physical phenomena, such as hopping of electrons through nanoparticles under the influence of magnetic fields, may also be possible using the aligned magnetic nanoparticles presented in this study. Acknowledgment. This study is partially supported by Human Frontier Science Program. The authors thank M. Yamane for her excellent technical assistance, S. Fujita for high-resolution TEM and EDS analyses, Dr. K. Ihara and K. Hasegawa for illustration, and Drs. S. Kawakami, K. Ajito, and K. Namba for discussion. Professional English proofreading by S. Nishida is also acknowledged.
FIGURE 5. The field dependence of magnetization (M-H) curve of nanoparticles formed inside the E106K mutant measured at 10 K.
periodical distance, which may be derived from the ordered helical structure of the virus whose helical pitch is about 2.3 nm. In Pt/Co nanoparticle formation, each particle appeared to grow larger after each sonication/reduction cycle (data not shown). In wt virus, Co2+ and PtCl42- ions are assumed to be supplied through two ends of the virus because there is no occasion on which three or more nanowires are observed within a single virus particle (Figure S1c in Supporting Information). In E106K mutant, Co2+ and PtCl42- ions may also be supplied through small gaps in the helical pitch, resulting in the simultaneous growth of many nanoparticles in a virus. Lattice fringes were observed in some particles (Figure 4a) indicating that those individual particles are crystalline and the lattice fringe distance of 0.2 nm indicates that the crystals are made of Pt, CoPt3, or CoPt. The possibility that they are made of Co is dismissed because it would not result in a lattice fringe distance of around 0.2 nm. Energy dispersive X-ray spectroscopy (EDS) analysis showed that the percentage of Co content ranged from 12 to 25%, which indicates that the collection of nanoparticles formed inside ToMV were composed mainly of Pt (Figure 4b). These results suggest that the particles are a mixture of crystalline Pt or CoPt3. It is also possible that some particles were amorphous Pt which was crystallized by the electron beam of TEM during observation. Because the diameter is smaller for particles formed with only Pt than those for Pt/Co (Figure 4a and Figure S2a in Supporting Information), it is possible that Pt forms the nucleation site and catalyzes the growth of Pt/Co nanoparticles on the surface of Pt. Using double or half amounts of (NH4)2Co(SO4)2 or K2PtCl4 solutions in nanoparticle forming conditions appeared to give similar results (data not shown). Magnetic properties of aligned nanoparticles have been confirmed by superconducting quantum interference device (SQUID) analysis (Figure 5). The M-H curve (field dependence of magnetization) showed a clear hysteresis loop with a coercive field (Hc) of 50 Oe at 10 K. This result indicates the presence of magnetic ordering in the one-dimensionally packed nanoparticles. The aligned nanoparticles produced in this study not only are promising components for bottom-up fabrication of other nanometer-scaled devices but also are intriguing subjects due to their electrical and magnetic properties. © 2010 American Chemical Society
Supporting Information Available. Details regarding the materials and methods used and supporting figures mentioned in the text. This material is available free of charge via the Internet at http://pubs.acs.org. REFERENCES AND NOTES (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24)
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