NANO LETTERS
Biological Routes to Metal Alloy Ferromagnetic Nanostructures
2004 Vol. 4, No. 6 1127-1132
Brian D. Reiss,†,‡ Chuanbin Mao,# Daniel J. Solis,§ Katherine S. Ryan,| Thomas Thomson,⊥ and Angela M. Belcher*,†,‡ Department of Materials Science and Engineering, DiVision of Biological Engineering, Department of Chemistry, and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, Hitachi San Jose Research Center, San Jose, California 95120, and Department of Chemistry and Biochemistry, Institute of Cellular and Molecular Biology, UniVersity of Texas at Austin, Austin, Texas 78712 Received January 30, 2004; Revised Manuscript Received March 10, 2004
ABSTRACT Magnetic nanoparticles have potential applications in high-density memory devices, but their complicated synthesis often requires high temperatures, expensive reagents, and postsynthesis annealing to achieve the desired magnetic properties. Current synthetic methods for magnetic nanoparticles often require post-synthetic modifications, suggesting that the practical application of magnetic nanoparticles will depend on the development of alternative synthetic strategies. We report a biological template to directly grow magnetic nanoparticles of desired material composition and phase under ambient conditions. A phage display methodology was adapted to identify peptide sequences that both specifically bind to the ferromagnetic L10 phase of FePt and control the crystallization of FePt nanoparticles using a modified arrested precipitation technique. TEM, electron diffraction, STEM, and X-ray diffraction all indicate these nanoparticles are composed of an FePt alloy with some degree of chemical ordering, and SQUID analysis shows these nanostructures are ferromagnetic at room temperature, possessing coercivities up to 1000 Oe.
Since the seminal work of Murray and colleagues,1 there has been a great deal of interest in using self-assembled films of hard-magnetic FePt nanoparticles for ultrahigh density memory devices. Nanoparticles of ferromagnetic materials are of importance due to their reduced sizes which can support only single magnetic domains, potentially leading to dramatic increases in storage density. However, the current post-synthesis annealing required to achieve the chemically ordered, high anisotropy ferromagnetic L10 phase leads to poor control over the spatial arrangement of nanoparticles through extensive particle aggregation.2 Additionally, since the particles are not prepared with the desired crystallinity, it makes organizing them on a surface with their magnetic easy axis aligned difficult, limiting their technological applications. Eliminating or reducing the high temperature annealing step would greatly simplify the production of magnetic nanoparticles suitable for use in device applications. To date, the most successful approach has been to lower the L10 phase transformation temperature through doping of the FePt nanoparticles with silver;3 yet annealing at temperatures * Corresponding author. E-mail:
[email protected]. † Department of Materials Science and Engineering. ‡ Division of Biological Engineering. § Department of Chemistry. | Department of Biology. ⊥ Hitachi San Jose Research Center. # University of Texas at Austin. 10.1021/nl049825n CCC: $27.50 Published on Web 04/30/2004
© 2004 American Chemical Society
>350 °C is still necessary. The need to remove the annealing process from the synthesis of high anisotropy magnetic nanoparticles suggests that a radically different approach be developed. Biological organisms have evolved the ability to direct the synthesis and assembly of crystalline inorganic materials under environmentally benign conditions with control over chemical composition and phase. Examples include the use of viruses expressing material-specific peptides to nucleate semiconducting nanoparticles,4,5 the use of porous protein crystals,6,7 modification of the iron storage protein ferritin,8,9 manipulation of bacteria and yeast to produce iron oxide10 and semiconducting nanoparticles,5,11,12 and selection of metal-specific polypeptides from combinatorial libraries.13 Most of these systems have focused on preparing materials composed of sulfides,8,11,12 calcium carbonate,14-16 silicon oxide,17 iron oxides,9,18 and noble metals,13,19-21 which are often similar to naturally abundant, biologically prepared inorganic materials. We have applied a peptide-based synthetic strategy for the synthesis of the technologically important magnetic material FePt, in which biological interactions control the nucleation of nanoparticles that have no isomorphous complement in nature. In addition, this biological strategy has several advantages over more recent chemical methods, including the potential synthesis of the desired high anisotropy ferromagnetic L10 crystalline phase
in aqueous conditions under ambient temperature, pressure, and atmosphere. Bacterial amplification of the “organic” virus templates makes the production of both the nucleating peptides and FePt nanoparticles easy and cost-effective. Phage display technology4,22 was applied to both annealed nanoparticle assemblies and thin films of L10 FePt,1 allowing for the rapid identification of peptides that selectively bind to the material of interest from a pool of 1010 unique sequences. Five rounds of screening with a dodecapeptide library yielded the dominant sequences for L10 FePt: HNKHLPSTQPLA, SVSVGMKPSPRP, VISNHRESSRPL, and KSLSRHDHIHHH. These sequences contain numerous amines, known to be excellent ligands for Pt, lysine, believed to be essential in the mechanism of the binding of HMG domain proteins to the Pt/DNA complex formed in cisplatinbased cancer therapies,23 and histidine, which is used extensively to bind Pt salts for studying protein activity24,25 and for the heavy metal staining of proteins to facilitate in X-ray crystallography.25 Basic local alignment search tool (BLAST) searches performed on these peptides showed that they all possess similarities to motifs found in naturally occurring proteins. For example, the metal-binding protein Fe (III)-coprogen receptor of Salmonella typhimurium contains the KHLPST26 amino acid motif identical to the central section of the FePt-specific dodecapeptide isolated in these experiments. The KHLPST motif can also be found in pilin, a fiber-forming protein found in Escherichia coli and numerous other microorganisms.27 Interestingly, the HNKHLPSTQPLA sequence was isolated in both the selection performed on FePt nanoparticle assemblies and FePt thin films and was therefore used exclusively for subsequent experiments. Initial nucleation experiments in which metal salt precursors were chemically reduced in the presence of the FePtspecific peptides, expressed as fusion proteins on the proximal tip of the virus (specifically the gP3 protein), produced a number of disordered phase FePt particles. Since only three to five copies of the gP3 protein are expressed on each phage, this approach does not provide a high enough concentration of peptide to compete with nonspecific precipitation effects. To circumvent this problem, the peptide concentration was significantly increased through the engineering of bacteriophage to express the desired peptide as a fusion to the gP8 protein, which has a copy number of ∼2700 per virus, and through the use of synthetically prepared peptide. It has been previously shown that the gP8 protein of the M13 bacteriophage can be modified to express high copy numbers of fusion proteins,28 and has been used to grow nanoparticles composed of II-VI semiconducting materials.29 FePt-specific sequences were cloned into a phagemid vector and expressed as fusions to the gP8 protein of the bacteriophage using a previously described technique.4 These engineered viruses were then used in particle nucleation experiments. To accomplish this, 1 mL of engineered phage (1012 phage/mL) was mixed with 1 mL of 0.075 M FeCl2 and 1 mL of 0.025 M H2PtCl6. This mixture was vortexed for 10 minutes to ensure mixing, and 1 mL of 0.1 M NaBH4 1128
Figure 1. TEM characterization of FePt nanoparticles prepared using engineered viral templates, including low resolution (a), high resolution (b), and selected area electron diffraction (c). Composite film of virus linked to FePt nanoparticles imaged using optical micrsocopy with a picture of the macroscopic film (inset) (d). Scale bars are 20 nm, 5 nm, and 20 mm in a, b, and c, respectively.
was added to reduce the metals forming the desired nanoparticles. Figure 1a and 1b are low-resolution and highresolution transmission electron microscope (TEM) images of FePt nanoparticles prepared using gP8 engineered phage as templates (all TEM images are taken using a JEOL 2010 TEM). These nanoparticles have an average diameter of 4.0 ( 0.6 nm, which is larger than the theoretically predicted minimum size of ∼2.5 nm required for thermal stability of L10 FePt nanoparticles. The lattice spacing of these particles as imaged by TEM is 0.22 and 0.26 nm and is in good agreement with the literature values of 0.219 nm for the (111) and 0.27 for the (110) planes of L10 FePt (PDF # 65-1051). Additionally, the selected area electron diffraction pattern (Figure 1c) shows rings corresponding to the (001), (110), (111), and (200) planes. Previous experiments with FePt nanoparticles have demonstrated that the (001) and (110) bands are not present in films prepared from preannealed, disordered FePt nanoparticles,1,30 and its presence here indicates that a significant percentage of nanoparticles are nucleated as the L10 phase of FePt. The ultimate goal of these experiments is to directly prepare nanoparticles composed of L10 FePt under ambient conditions, which is a challenge as this is not the kinetically favorable phase of FePt. Since TEM characterization indicates that some chemical ordering is present in these samples, it is reasonable to believe that the biological approach to materials synthesis can ultimately overcome this kinetic barrier and be used to directly prepare the L10 phase of FePt. Previously it has been shown that high concentrations of bacteriophage linked to ZnS nanoparticles can be converted into hybrid films possessing long-range order by slowly evaporating the solvent.5 Preparing similar films from viruses conjugated to magnetic nanoparticles would also be of interest due to their potential for data storage. Figure 1d is one such film prepared from viruses conjugated to FePt Nano Lett., Vol. 4, No. 6, 2004
Figure 3. SQUID characterization of similar nanoparticles taken at 300 K (solid line) and 5 K (dashed line).
Figure 2. STEM image of FePt nanoparticles prepared using engineered viral templates (a). Fe (b) and Pt (c) map of identical nanoparticles taken using high angle annular dark field STEM. Scale bar in A is 350 nm.
nanoparticles prepared as described above and imaged using optical microscopy. These films have a nanoparticle density of approximately 6 × 1012 nanoparticles/in.2, opening the possibility of applications based on this technology. Nano Lett., Vol. 4, No. 6, 2004
Scanning transmission electron microscopy (STEM) was used to obtain chemical information of the nanoparticlevirus system. Figure 2a is a STEM image of an FePt mineralized phage that can only be imaged due to the heavier elements associated with the attached nanoparticles. High angle annular dark field (HAADF) STEM elemental mapping of Fe (2b) and Pt (2c) confirms that the nanoparticles are composed of an alloy of Fe and Pt. Control experiments involving phage expressing a random peptide, wild-type phages that do not express any insert, and without phage produce only noncrystalline and polycrystalline particles of a disordered phase of FePt. Superconducting quantum interference device (SQUID) magnetometry of the as-prepared FePt particles at 5 K and 300 K showed coercivities of 1350 and 200 Oe respectively (Figure 3). Although the room temperature coercivity is much less than might be expected for a sample containing only L10 FePt nanoparticles, it does provide evidence of the existence of some high anisotropy FePt nanoparticles. The particles prepared in these experiments are also close to the superparamagnetic limit of L10 FePt, and this might also contribute to low coercivity observed in these experiments. The low value found for the squareness, S ) mr/ms ) 0.09, compared with an ideal value of 0.48 expected for a fully chemically ordered, random ensemble of nanoparticles, is again indicative of only partial L10. Additionally, X-ray diffraction data (not shown) indicate that while the biological templates have some ability to nucleate ordered FePt, this process must be optimized to prepare fully ordered L10 FePt as desired. It has also been shown that high temperature annealing of biologically prepared FePt and CoPt samples also leads to aggregation of the nanoparticles,31 which could be used to tune the magnetic properties of the nanoparticles. As a control experiment, FePt nanoparticles were grown using a virus engineered to express a peptide on its g8P protein that was not specific to FePt (the sequence SPPRNYYSSMSS was isolated from a selection on an unrelated material). The nanoparticle synthesis was carried out in a fashion identical to the procedure described above. Figure 4 shows the TEM characterization of the resulting 1129
Figure 4. Low resolution TEM images of the unique FePt nanostructures grown using nonspecific peptides (a and b) and a selected area electron diffraction image of the nanoparticles in a (c). Two batches of nanoparticles (d) grown using an FePt-specific peptide (left) and a nonspecific peptide (right).
nanostructures. Two types of structures can be found in these samples. Figure 4a shows aggregates of spherical nanoparticles, while Figure 4b shows some more filamentous nanoparticles. It is not clear why these different nanostructures form in the control experiment, but it is important to point out that neither of these nanostructures is composed of the crystalline material shown in Figure 1a. Figure 4c is a selected area electron diffraction image of one of the structures shown in Figure 4a. The only band that can be clearly seen in this image roughly correlates to the (111) plane of FePt, but this plane is not indicative of L10 FePt, suggesting that very little chemical ordering is obtained in these samples when a nonspecific peptide is used to control the nucleation. Figure 4d shows two vials in which FePt nanoparticles were nucleated. The vial on the left contained virus expressing the FePt sequence, while the vial on the right contained a control sequence. After several minutes, the nanoparticles in the FePt sample are still dispersed in solution, but the nanoparticles in the control experiment have aggregated and precipitated from solution. This result indicates that the FePt-specific peptide is crucial for nucleating and stabilizing these nanoparticles in solution. Previously, it has been shown that, by varying reaction conditions,32-34 nanoparticles of various shapes and sizes can be prepared. We have also studied the gP8 system described above to determine if this system possesses similar tunability. By heating the sample to 60 °C and tripling the Fe2+ concentration, it was found that highly anisotropic FePt nanostructures could be prepared. TEM images of these samples show crystalline domains in these structures having a lattice spacing of 0.21 nm (Figure 5a), corresponding to the (111) plane of FePt, that vary in length from 70 to 800 nm due to shearing of the viral templates from heating and chemical treatment (Figure 5b). Additionally, crystalline sheets growing off the nanowire surface extend for tens of nanometers with a lattice spacing of 0.26 nm (Figure 5c), 1130
Figure 5. Low-resolution TEM images of FePt nanowires grown using engineered viral templates (a). High-resolution image of an individual nanoparticle (b), and a high-resolution image of an FePt “sheet” growing from a nanowire (c) with a selected area electron diffraction pattern of a nanowire (d). Scale bars are 100, 10, and 5 nm (a, b, and c, respectively). Nano Lett., Vol. 4, No. 6, 2004
Figure 6. X-ray diffraction of FePt nanowires grown using engineered viral templates (a) and SQUID characterization at 300 K of similar nanowires (b).
that corresponds to the (110) plane of FePt. The inset in Figure 5C is a selected area electron diffraction pattern of the FePt nanoparticles, showing bands corresponding to the (001), (110), (111), (002), (201), (112), and (003) planes of FePt. Figure 6a is an XRD pattern of the as-prepared anisotropic FePt nanoparticles with the (111) peak at 40.5° being the most dominant feature. The (111) peak is broadened due to the small size of these particles and obscures several other important diffraction peaks. For example, the (200) peak at 47° and (110) peak at 33° appear as shoulders on the (111) peak. Additionally, the (001) peak appears as a very weak peak at 23°, indicating the presence of L10 FePt. The peak at 35° correlates to iron oxide and is attributed to the large amounts of Fe used in the synthesis. Figure 6b is a hysteresis loop taken of this sample at 300 K and shows a coercivity of 1000 Oe, which is expected due to the larger particle size and increased anisotropy of these nanostructures. Synthetically prepared FePt-specific dodecapeptides were also used to nucleate nanoparticles with an average diameter of 4.1 + 0.6 nm, and a lattice spacing 0.22 nm that agrees well with the literature value of 0.2197 nm for the (111) facet of FePt (Figure 7a and 7b). Selected area electron diffraction (Figure 7c) suggests that these nanoparticles contain some chemical ordering. In particular, the presence of bands correlating to the (110) and (001) planes of FePt indicate that L10 FePt may be formed in these experiments. SQUID characterization at 300 K (Figure 7d) shows some Nano Lett., Vol. 4, No. 6, 2004
Figure 7. Low (a) and high (b) resolution images of FePt nanoparticles grown using synthetically prepared peptide as a template with diffraction characterization, including X-ray diffraction (c) and selected area electron diffraction (d). SQUID characterization of similar particles (e) taken at 300 K, and 1 µm × 1 µm AFM images of a single monolayer of particles (g) and 5 layers of particles (h) deposited onto a Si wafer. Scale bars are 10 and 5 nm in a and b.
hysteresis at room temperature with Hc ) 670 Oe. The presence of ferromagnetic particles at room temperature in these samples, which have not undergone an annealing step,1,35 represents a potential synthetic strategy for preparing ferromagnetic particles dispersed in solution rather than immobilized on a surface. The ferromagnetic nature of these nanoparticles suggests they have potential applications in data storage technology. To test this idea, films of the nanoparticles were prepared by modifying Si wafers with an amine-functionalized silane. The wafers were then immersed in a solution of the nanoparticles for 30 min. After rinsing, the wafers were submerged in a solution of 0.5% poly(vinylpyrrolidone) for 15 min. The wafers were then rinsed with water and placed back in the nanoparticle solution. This process was repeated several times to yield multilayers of the magnetic particles. Figure 7 shows AFM images of such multilayer samples with 1 (Figure 7e) and 5 (Figure 7f) layers of particles. These AFM measurements indicate the films have less than 1-nm root-mean-square variation in height over areas as large as 1131
5 µm × 5 µm. These low values of surface roughness are essential for performing static read/write tests, which are in progress. The ability to fabricate smooth films from our ferromagnetic particles suggests that these particles could act as viable alternatives to the FePt nanoparticles prepared using traditional synthetic techniques to form high-density arrays of nanoparticles that do not require an annealing step for practical device applications. Previously it has been shown that polyanionic proteins isolated from abalone shells that possess a high affinity for CaCO3 can be used to control the crystallization of CaCO3 crystals grown in vitro.15,16 Our research group has reported peptides that can control the crystallization of ionic solids such as II-IV semiconductors, which are isomorphic to naturally occurring biominerals.4 The peptides selected in these experiments bind specifically to magnetic materials that are radically different than previously investigated biominerals and have been shown to exhibit similar control over the crystallization of magnetic nanostructures. The application of this technology to radically different materials and different chemical reactions suggests that peptides have the potential to fabricate a variety of inorganic materials that do not naturally coexist with biological systems. Additionally, the protocol is very robust and can be applied to alternative magnetic materials. Currently, we are investigating the synthesis of CoPt, SmCo5, and Co. The development of biological routes for the synthesis of magnetic materials not only provides a “green” synthetic approach but also can overcome issues with traditional methods such as hightemperature annealing. Acknowledgment. We gratefully acknowledge Dr. Chris Murray for his insightful comments and the Air Force Office of Scientific Research, NSF, and IBM Faculty Partnership for their support. We would like to acknowledge the use of the core microscopy facilities in the Texas Materials Institute, Center for Nano- and Molecular Science and Technology, and the Institute of Cellular and Molecular Biology at the University of Texas at Austin. We also acknowledge the use of core microscopy and SQUID facilities in the Center for Materials Science and Engineering at the Massachusetts Institute of Technology. We thank Dr. Ronald Daas for his assistance with SQUID measurements. References (1) Sun, S.; Murray, C. B.; Weller, D.; Folks, L.; Moser, A. Science 2000, 287, 1989-1992. (2) Dai, Z. R.; Sun, S.; Wang, Z. L. Nano Lett. 2001, 1, 443-447. (3) Kang, S.; Harrell, J. W.; Nikles, D. E. Nano Lett. 2002, 2, 10331036. (4) Mao, C.; Flynn, C. E.; Hayhurst, A.; Sweeney, R.; Qi, J.; Georgiu, G.; Iverson, B.; Belcher, A. M. Proc. Nat. Acad. Sci. U.S.A. 2003, 100, 6946-6951.
1132
(5) Lee, S. W.; Mao, C.; Flynn, C. E.; Belcher, A. M. Science 2002, 296, 892-895. (6) Douglas, T.; Dickson, D. P. E.; Betteridge, S.; Charnock, J.; Garner, C. D.; Mann, S. Science 1995, 269, 54-57. (7) Douglas, T.; Young, M. Nature 1998, 393, 152-155. (8) Wong, K. K. W.; Mann, S. AdV. Mater. 1996, 8, 928-933. (9) Douglas, T.; Stark, V. T. Inorg. Chem. 2000, 39, 1828-1830. (10) Sakaguchi, T.; Burgess, J. G.; Matsunga, T. Nature 1993, 365, 4749. (11) Dameron, C. T.; Reese, R. N.; Mehra, R. K.; Kortan, A. R.; Carroll, P. J.; Steigerwald, M. L.; Brus, L. E.; Winge, D. R. Nature 1989, 338, 596-597. (12) Kowshik, M.; Vogel, W.; Urban, J.; Kulkarni, S. K.; Paknikar, K. M. AdV. Mater. 2002, 14, 815-818. (13) Brown, S.; Sarikaya, M.; Johnson, E. J. Mol. Biol. 2000, 299, 725735. (14) Zaremba, C. M.; Belcher, A. M.; Fritz, M.; Li, Y.; Mann, S.; Hansma, P. K.; Morse, D. E.; Speck, J. S.; Stucky, G. D. Chem. Mater. 1996, 8. (15) Belcher, A. M.; Wu, X. H.; Christensen, R. J.; Hansma, P. K.; Stucky, G. D.; Morse, D. E. Nature 1996, 381, 56-58. (16) Falini, G.; Albeck, S.; Weiner, S.; Addadi, L. Science 1996, 271, 67-69. (17) Fowler, C. E.; Shenton, W.; Stubbs, G.; Mann, S. AdV. Mater. 2001, 13, 1266-1269. (18) Shenton, W.; Mann, S.; Colfen, H.; Bacher, A.; Fischer, M. AdV. Mater. 2001, 40, 442-445. (19) Dujardin, E.; Peet, C.; Stubbs, G.; Culver, J. N.; Mann, S. Nano Lett. 2002. (20) Naik, R. R.; Stringer, S. J.; Agarwal, G.; Jones, S. E.; Stone, M. O. Nature Mater. 2002, 1, 169-172. (21) Mukherjee, P.; Ahmad, A.; Mandal, D.; Senapati, S.; Sainkar, S. R.; Khan, M. I.; Parishcha, R.; Ajaykumar, P. V.; Alam, M.; Kumar, R.; Sastry, M. Nano Lett. 2001, 1, 515-519. (22) Whaley, S. R.; English, D. S.; Hu, E. L.; Barbara, P. F.; Belcher, A. M. Nature 2000, 405, 665-668. (23) Kane, S. A.; Lippard, S. J. Biochemistry 1996, 35, 2180-2188. (24) Mooney, B. P.; David, N. R.; Thelen, J. J.; Miernyk, J. A.; Randall, D. D. Biochem. Biophys. Res. Commun. 2000, 267, 500-503. (25) Lupinkova, L.; Metz, J. G.; Diner, B. A.; Vass, I.; Komenda, J. Biochim. Biophys. Acta 2002, 1554, 192-201. (26) McClelland, M.; Sanderson, K. E.; Spieth, J.; Clifton, S. W.; Latreille, P.; Courtney, L.; Porwollik, S.; Ali, J.; Dante, M.; Du, F.; Hou, S.; Layman, D.; Leonard, S.; Nguyen, C.; Scott, K.; Holmes, A.; Grewal, N.; Mulvaney, E.; Ryan, E.; Sun, H.; Florea, L.; Miller, W.; Stoneking, T.; Nhan, M.; Waterston, R.; Wilson, R. K. Nature 2001, 413, 852-856. (27) Parge, H. E.; Forest, K. T.; Hickey, M. J.; Christensen, D. A.; Getzoff, E. D.; Tainer, J. A. Nature 1995, 378, 32-38. (28) Malik, P.; Terry, T. D.; Gowda, L. R.; Langara, A.; Petukhov, S. A.; Symmons, M. F.; Welsh, L. C.; Marvin, D. A.; Perham, R. N. J. Mol. Biol. 1996, 260, 9-21. (29) Flynn, C. E.; Mao, C.; Hayhurst, A.; Williams, J. L.; Georgiou, G.; Iverson, B.; Belcher, A. M. J. Mater. Chem. 2003, 13, 2414-2421. (30) Dai, Z. R.; Sun, S.; Wang, Z. L. Nano Lett. 2001, 1, 443-447. (31) Mao, C.; Solis, D. J.; Reiss, B. D.; Kottmann, S. T.; Sweeney, R.; Hayhurst, A.; Georgiu, G.; Iverson, B.; Belcher, A. M. Science 2004, 303, 213-217. (32) Puntes, V. F.; Krishnan, K. M.; Alivisatos, A. P. Science 2001, 291, 2115-2117. (33) Manna, L.; Scher, E. C.; Alivisatos, A. P. J. Am. Chem. Soc. 2000, 122, 12700-12706. (34) Jun, Y.-w.; Jung, Y.-y.; Cheon, J. J. Am. Chem. Soc. 2002, 124, 615619. (35) Warne, B.; Kasyutich, O. I.; Mayes, E. L.; Wiggins, J. A. L.; Wong, K. K. W. IEEE Trans. Magn. 2000, 36, 3009-3011.
NL049825N
Nano Lett., Vol. 4, No. 6, 2004
