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Iron and Cobalt Oxide and Metallic Nanoparticles Prepared from Ferritin Hazel-Ann Hosein,† Daniel R. Strongin,*,† Mark Allen,‡ and Trevor Douglas‡ Department of Chemistry, Temple University, Philadelphia, Pennsylvania 19122, and Department of Chemistry & Biochemistry, Montana State University, Bozeman, Montana 59717 Received April 7, 2004. In Final Form: August 11, 2004 Metallic Fe and Co and Fe- and Co-based oxide nanoparticles were prepared by a novel method utilizing the biologically relevant protein ferritin. In particular, iron and cobalt oxyhydroxide nanoparticles were assembled within horse spleen and Listeria innocua derived ferritin, respectively, in the aqueous phase. Ferritin containing either Fe or Co oxide was transferred and dried on a SiO2 support where the protein shell was removed during exposure to a highly oxidizing environment. It was also shown that the metal oxide particles could be reduced to the respective metal by heating in hydrogen. X-ray photoelectron spectroscopy was used to characterize the composition of the particles and atomic force microscopy was used to characterize the size of the nanoparticles. Depending on the Fe or Co loading and/or type of ferritin used, metallic and oxide nanoparticles could be produced within a range of 20-60 Å.
1. Introduction Metallic and metal oxide nanoparticles have become an area of growing interest and importance in a wide range of fundamental studies and technological applications, due to their unique optical, electronic, magnetic, chemical, and mechanical properties.1-6 Furthermore, nanoparticles, often metal oxides, are increasingly being associated with important environmental processes occurring in soils and the atmosphere.7 Hence, synthetic routes to produce nanoparticles for technological purposes and for model systems for environmental studies are needed. Research has shown that the properties of the nanoparticles are often different from their bulk counterparts.8,9 With regard to metallic nanoparticles, for example, the conduction band, which is present in bulk metal, is absent in nanometal, where instead there are discrete states at the band edge. At the nanoscale level, transition metals, like Co in particular, show magnetic behavior that is dependent on size.10 Other nanosized materials, such as TiO2, show specific enhancements in redox properties,11 while nanoclusters of gold have been shown to exhibit a catalytic activity that cannot be duplicated by bulk gold particles.12-15 Au on TiO2 is an interesting case of how unique catalytic activity can be obtained if the spatial * To whom correspondence may be addressed. E-mail: dstrongi@ temple.edu. † Temple University. ‡ Montana State University. (1) Edelstein, A. S.; Cammarata, R. C. Nanomaterials: Synthesis, Properties and Application; Institute of Physics Publishing: London, 1996. (2) Hayashi, C.; Uyeda, R.; Tasaki, A. Ultra-fine particles: Exploratory Science and Technology; Noyes Publications: Norwich, NY, 1997. (3) Hadjipanayis G. C.; Prinz, G. A. Science and Technology of Nanostructured Magnetic Materials; Plenum Press: New York, 1991. (4) Zhang W.; Wang, C.; Lien, H. Catal. Today 1998, 40, 387. (5) Zhang W.; Wang, C. Environ. Sci. Technol. 1997, 31, 2154. (6) Poizot, P.; Laruelle, S.; Grugeon, S.; Dupont, L.; Tarascon, J.-M. Nature 2000, 407, 496. (7) See articles in: Banfield, J. F., Navrotsky, A., Eds. Nanoparticles and the Environment. In Rev. Mineral. Geochem. 2001, 44. (8) Schmid, G. Clusters and Colloids; VCH: Weinheim, 1994. (9) Halperin, W. P. Rev. Mod. Phys. 1986, 58, 533. (10) Puntes, V. F.; Drishnan, K. M.; Alivisatos, A. P. Nature (London) 2001, 291, 2115. (11) Hoffmann, M. R.; Martin, S. T.; Choi, W.; Bahneman, D. W. Chem. Rev. 1995, 95, 69.
dimension of the catalytic particle is brought into the nanoscale regime. Prior studies have shown that nanoparticles of Au supported on the TiO2 surface exhibit a high activity toward CO oxidation, chemistry that is not experimentally observable on bulk Au.12,13,15-19 The electronic structure20 and ultimately the reactivity of Au nanoparticles show a strong dependence on size. Au particles with a diameter of approximately 2 nm show the largest CO oxidation behavior.21 Such a size dependence might be expected to occur in other metallic systems. This result emphasizes the need to synthesize nanoparticles with well-defined sizes, so that surface reactivities can be correlated to size. Currently, many techniques are being used for the preparation of submicrometer to nanometer sized particles. These include chemical vapor deposition,22 laser vaporization,23 electron beam lithography,24 and various colloidal syntheses such as the normal/reverse micelle method, double emulsion, and Langmuir monolayer techniques25-29 as well as a range of organometallic techniques.30 However, the preparation of uniform nano(12) Haruta, M.; Tsubota, S.; Kobayashi, T.; Kageyama, H.; Genet, M. J.; Delmon, B. J. Catal. 1993, 144, 175. (13) Valden, M.; Goodman, D. W. Isr. J. Chem. 1998, 38, 285. (14) Haruta, M.; Uphade, B. S.; Tsubota, S.; Miyamoto, A. Res. Chem. Intermed. 1998, 24, 329. (15) Haruta, M. Stud. Surf. Sci. Catal. 1997, 110, 123. (16) Rodriguez, J. A.; Perez, M.; Jirsak, T.; Evans, J.; Hrbek, J.; Gonzalez, L. Chem. Phys. Lett, 2003, 378, 526. (17) Takaoka, G. H.; Hamano, T.; Fukushima, K.; Jiro, M.; Yamada, I. Nucl. Instrum. Methods Phys. Res., Sect. B 1997, 121, 503. (18) Bondzie, V. A.; Parker, S. C.; Campbell, C. T. Catal. Lett. 1999, 63, 143. (19) Haruta, M.; Tsubota, S.; Kobayashi, T.; Ueda, A.; Sakurai, H.; Ando, M. Stud. Surf. Sci. Catal. 1993, 75, 2657. (20) Chusuei, C. C.; Lai, X.; Luo, K.; Goodman, D. W. Top. Catal. 2001, 14, 71. (21) Lai, X.; Clair, T. P. S.; Valden, M.; Goodman, D. W. Prog. Surf. Sci. 1998, 59, 25. (22) Choi, C. J.; Tolochko, O.; Kim, B. K. Mater. Lett. 2002, 56, 289. (23) El-Shall, M. S.; Slack, W.; Vann, W.; Kane, D.; Hanley, D J. Phys. Chem. 1994, 98, 3067. (24) Eppler, A. S.; Zhu, J.; Anderson, E.; Somorjai, G. A. Top. Catal. 2000, 13, 33. (25) Fendler, J. H.; Meldrum, F. C. Adv. Mater. 1995, 7, 607. (26) Khomutov G. B. Colloids Surf., A 2002, 202, 243. (27) Pileni, M. P. J. Phys. Chem. 1993, 97, 6961. (28) Taleb, A.; Petit, M. P.; Pileni, M. P. Chem Mater. 1997, 9, 950. (29) Calvin, S.; Carpenter, E. E.; Harris, V. G. Phys. Rev., B: Condens. Matter Mater. Phys. 2003, 68, 3, 0334111.
10.1021/la0491100 CCC: $27.50 © 2004 American Chemical Society Published on Web 10/08/2004
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sized, monodispersed transition metal based particles still remains a significant challenge. Recently, it has been shown that the biologically relevant protein ferritin could be used to prepare monodispersed films of iron oxyhydroxide nanoparticles.31,32 In particular, horse spleen ferritin containing 80 Å diameter particles of the oxyhydroxide was supported on a substrate and the protein shell was removed by a reactive oxygen environment. It was shown using X-ray diffraction that the oxyhydroxide could be reduced to iron metal by reduction in hydrogen at elevated temperatures. It was not shown, however, whether the well-defined iron oxyhydroxide particles retained their nanoparticle morphology after reduction. The present contribution builds upon this earlier work and shows the effect of metal loading and ferritin-type on the ultimate nanoparticle size. Ferritin derived from two sources, horse spleen and Listeria innocua, was used to assemble metal oxides of varying sizes and compositions. Horse spleen ferritin is composed of 24 polypeptide subunits that fold to produce a cage structure, having an outer diameter of 120 Å and an inner core diameter of 80 Å, and can accommodate up to 4500 Fe atoms as a mineral of ferrihydrite.33 Listeria ferritin-like protein (LFLP) has a similar cage structure, but the inner core diameter is only about 56 Å and has been shown to accommodate only 500 atoms of Fe or Co as oxides.34-36 In this work, the versatility of these ferritin proteins was exhibited in two ways. First, it is shown that by individually assembling iron oxyhydroxide particles within horse spleen ferritin with two different Fe loadings, supported iron oxyhydroxide nanoparticles with nominal 25 and 60 Å diameters could be produced after the removal of the protein shell. It is also shown for the larger size particles that the reduction of the oxyhydroxide particle to Fe metal could be carried out. Second, by using the LFLP, small cobalt oxyhydroxide particles in the 30 Å range could be produced. The reduction of the cobalt nanoparticle to the corresponding metal is also described. Atomic force microscopy was used as the primary sizing tool for the characterization of these nanostructures and provided a range of information regarding surface topography and surface texture, while X-ray photoelectron spectroscopy (XPS) was used mainly to determine surface composition. 2. Materials and Methods 2.1. Preparation of Samples. Horse spleen ferritin was prepared with Fe atom loading of 100 and 2500 as previously described.37,38 Specifically, 100 Fe loaded ferritin was prepared by the addition of 0.04 mL of a deaerated solution of ferrous ammonium sulfate [(NH4)2Fe(SO4)‚6H2O] (10 mg/mL) to 5 mg of apoferritin (2.5 × 10-6 M, Sigma) in 20 mL of MES at pH 6.5 (0.1 M) followed by air oxidation. For the 2500 Fe loaded ferritin, five 0.2 mL aliquots of the ferrous ammonium sulfate solution were added with 1 h time intervals between additions. The remineralized ferritin was dialyzed into a 0.1 M tris buffer at a pH of 8.5 (30) Rockenberger, J.; Scher, E. C.; Alivisatos, A. P. J. Am. Chem. Soc., 1999, 121, 11595. (31) Hikono, T.; Yukiharu, U.; Fuyuki, T.; Yamashita, I. Jpn. J. Appl. Phys. 2003, 42, 398. (32) Furuno, T.; Sasabe, H.; Ikegami, A. Ultramicroscopy 1998, 70, 125. (33) Bozzi, M: Mignogna, G.; Stefanini, S.; Barra, D.; Longhi, C.; Valenti, P.; Chiancone, E. J. Biol. Chem. 1997, 272, 325920. (34) Stefanini, S.; Cavall, S.; Benedetaa, M.; Chiancone, E. Biochem. J. 1999, 338, 71. (35) Allen, M.; Willits, D.; Mosolf, J.; Young, M.; Douglas, T. Adv. Mater. 2002, 14, 1562. (36) Gider, S.; Awschalom, D. D.; Douglas, T.; Wong, K.; Mann, S.; Cain, G. J. Appl. Phys. 1996, 79, 5324. (37) Wong, K. K. W.; Douglas, T.; Gider, S.; Awschalom, D. D.; Mann, S. Chem. Mater. 1998, 10, 279. (38) Allen, M.; Willits, D.; Young, M.; Douglas, T. Inorg. Chem. 2003, 42, 6300.
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Figure 1. N 1s and C 1s XPS data of 2500 Fe loaded ferritin before (a) and after (b) UV-ozone treatment at 373 K for 60 min with O2 < 5 psi. Both the carbon and nitrogen associated with the protein shell are removed with the ozone treatment. for storage, but just prior to use it was dialyzed into purified water (18 MΩ resistivity) to remove any salts. Listeria innocua ferritin-like protein was expressed and purified from an E. coli expression system as described earlier36 and then mineralized so that there were 400 Co atoms per protein molecule.38 Size exclusion chromatography, dynamic light scattering, and transmission electron microscopy were utilized to assess the purity of the ferritin solutions. Atomic force microscopy (AFM) samples were prepared by dropping 20 µL of 0.10 mg/mL aqueous ferritin solutions on a 1 cm2 silicon wafer. The wafers were placed in a vacuum desiccator for approximately 10 min to evaporate the water and then UV-ozone treated (Novascan) for 60 min at 373 K under oxygen (
