Photochemical mineralization of europium, titanium, and iron oxyhydroxide nanoparticles in the ferritin protein cage

Article abstract of DOI:10.1021/ic701740q

The Fe storage protein ferritin was used as a size-constrained reaction vessel for the photoreduction and reoxidation of complexed Eu, Fe, and Ti precursors for the formation of oxyhydroxide nanoparticles. The resultant materials were characterized by dynamic light scattering, gel electrophoresis, UV-vis spectroscopy, and transmission electron microscopy. The photoreduction and reoxidation process is inspired by biological sequestration mechanisms observed in some marine siderophore systems.

Full text of DOI:10.1021/ic701740q

Inorg. Chem. 2008, 47, 2237-2239
Photochemical Mineralization of Europium, Titanium, and Iron
Oxyhydroxide Nanoparticles in the Ferritin Protein Cage
Michael T. Klem,†,‡ Jesse Mosolf,†,‡ Mark Young,*,‡,§ and Trevor Douglas*,†,‡
Department of Chemistry & Biochemistry, Department of Plant Sciences, and Center for
BioInspired Nanomaterials, Montana State UniVersity, Bozeman, Montana 59717
Received September 4, 2007
The Fe storage protein ferritin was used as a size-constrained
reaction vessel for the photoreduction and reoxidation of complexed
Eu, Fe, and Ti precursors for the formation of oxyhydroxide
nanoparticles. The resultant materials were characterized by
dynamic light scattering, gel electrophoresis, UV–vis spectroscopy,
and transmission electron microscopy. The photoreduction and
reoxidation process is inspired by biological sequestration mech-
anisms observed in some marine siderophore systems.
macromolecular templates,14 and evolved molecular interac-
tions15 to exert synthetic control over crystal morphology,
phase, and orientation. In particular, protein cage architec-
tures having high symmetry have been shown to act as
constrained reaction environments for the synthesis and
encapsulation of inorganic and organic nanomaterials.5,8,10–12,16
Using a biomimetic approach to materials synthesis, we
have developed a photochemical approach to generate
transient reduced metal species from an extremely stable pool
of chelated high-oxidation-state metal ions. The efficacy of
this approach was demonstrated by the spatially selective
mineralization of the spherical Fe storage protein cage
ferritin, which resulted in the formation of a stable metal
oxyhydroxide nanoparticles encapsulated within the protein
cage as potential precursors to other oxide materials.
Biomimetic approaches to materials chemistry provide new
avenues for the synthesis and assembly of nanomaterials.1–6
There is growing interest in materials chemistry to take
advantage of the physical and chemical properties of bio-
molecules for development of the next generation of nano-
scale materials.7 Bioinspired approaches to materials syn-
thesis have utilized well-defined protein architectures,8–13
Ferritins consist of 24 protein subunits that self-assemble
into a cagelike architecture with an exterior diameter of 12
nm and an interior diameter of 8 nm in which a hydrated
ferric oxide/phosphate is mineralized. Ferritins have evolved
to sequester Fe in ViVo, but the protein has been used as a
template in synthetic reactions with various metal ions, which
results in the formation of inorganic nanoparticles constrained
exclusively within the protein cage architecture. Materials
such as Fe3O4,13,17–21 Co3O4,8,22 Mn3O4,23–25 CoPt,26 Pd,27
* To whom correspondence should be addressed. E-mail: myoung@
montana.edu (M.Y.), tdouglas@chemistry.montana.edu (T.D.).
†
Department of Chemistry & Biochemistry.
Department of Plant Sciences.
Center for BioInspired Nanomaterials.
‡
§
(1) Lee, S. W.; Mao, C. B.; Flynn, C. E.; Belcher, A. M. Science 2002,
296, 892.
(2) Mann, S. Biomimetic Materials Chemistry; VCH: New York, 1996.
(3) Mann, S.; Archibald, D. D.; Didymus, J. M.; Douglas, T.; Heywood,
B. R.; Meldrum, F. C.; Reeves, N. J. Science 1993, 261, 1286.
(4) Naik, R. R.; Stringer, S. J.; Agarwal, G.; Jones, S. E.; Stone, M. O.
Nat. Mater. 2002, 1, 169.
(14) Shenton, W.; Douglas, T.; Young, M.; Stubbs, G.; Mann, S. AdV.
Mater. 1999, 11, 253.
(15) Allen, M.; Willits, D.; Mosolf, J.; Young, M.; Douglas, T. AdV. Mater.
2002, 14, 1562.
(16) McMillan, R. A.; Paavola, C. D.; Howard, J.; Chan, S. L.; Zaluzec,
N. J.; Trent, J. D. Nat. Mater. 2002, 1, 247.
(5) Uchida, M.; Klem, M. T.; Allen, M.; Suci, P.; Flenniken, M.; Gillitzer,
E.; Varpness, Z.; Liepold, L. O.; Young, M.; Douglas, T. AdV. Mater.
2007, 19, 1025.
(17) Bulte, J. W. M.; Douglas, T.; Mann, S.; Frankel, R. B.; Moskowitz,
B. M.; Brooks, R. A.; Baumgarner, C. D.; Vymazal, J.; Frank, J. A.
InVest. Radiol. 1994, 29, S214.
(6) Whaley, S. R.; English, D. S.; Hu, E. L.; Barbara, P. F.; Belcher,
A. M. Nature 2000, 405, 665.
(18) Gider, S.; Awschalom, D. D.; Douglas, T.; Mann, S.; Chaparala, M.
Science 1995, 268, 77.
(7) Niemeyer, C. M. Angew. Chem., Int. Ed. 2001, 40, 4128.
(8) (a) Allen, M.; Willits, D.; Young, M.; Douglas, T. Inorg. Chem. 2003,
42, 6300. (b) Klem, M. T.; Willits, D.; Young, M.; Douglas, T. J. Am.
Chem. Soc. 2003, 125, 10806. (c) Hosein, H. A.; Strongin, D. R.;
Allen, M.; Douglas, T. Langmuir 2004, 20, 10283.
(19) Gider, S.; Awschalom, D. D.; Douglas, T.; Wong, K.; Mann, S.; Cain,
G. J. Appl. Phys. 1996, 79, 5324.
(20) Pankhurst, Q. A.; Betteridge, S.; Dickson, D. P. E.; Douglas, T.; Mann,
S.; Frankel, R. B. Hyperfine Interact. 1994, 91, 847.
(21) Wong, K. K. W.; Douglas, T.; Gider, S.; Awschalom, D. D.; Mann,
S. Chem. Mater. 1998, 10, 279.
(9) Douglas, T.; Dickson, D. P. E.; Betteridge, S.; Charnock, J.; Garner,
C. D.; Mann, S. Science 1995, 269, 54.
(10) Douglas, T.; Strable, E.; Willits, D.; Aitouchen, A.; Libera, M.; Young,
M. AdV. Mater. 2002, 14, 415.
(22) Resnick, D. A.; Gilmore, K.; Idzerda, Y. U.; Klem, M. T.; Allen, M.;
Douglas, T.; Young, M.; Arenholz, E. J. Appl. Phys. 2006, 99, 08Q501.
(23) Mackle, P.; Charnock, J. M.; Garner, C. D.; Meldrum, F. C.; Mann,
S. J. Am. Chem. Soc. 1993, 115, 8471.
(11) Douglas, T.; Young, M. Nature 1998, 393, 152.
(12) Mann, S.; Ozin, G. A. Nature 1996, 382, 313.
(13) Meldrum, F. C.; Heywood, B. R.; Mann, S. Science 1992, 257, 522.
(24) Mann, S. Nature 1991, 349, 285–286.
10.1021/ic701740q CCC: $40.75  2008 American Chemical Society
Inorganic Chemistry, Vol. 47, No. 7, 2008 2237
Published on Web 02/29/2008

COMMUNICATION
Figure 1. Summary of the photochemical reaction cycle of M(III)L [M )
Eu, Fe, or Ti (as a IV salt); L ) citrate] and ferritin (Fn) to produce
oxyhydroxides in the ferritin protein cage. The metal citrate salts are
illuminated with a xenon arc lamp to reduce a small amount of the chelated
metal precursor, and this subsequently undergoes a reoxidation to form a
metal oxyhydroxide inside the protein cage.
Ag,28 Cu,29,30 CdS,31 CdSe,32 and ZnSe33 have been
synthesized within protein cages. The in Vitro mineralization
of ferritin is usually accomplished by a metal ion oxidative
hydrolysis polymerization. Typically, a low-oxidation-state
metal ion is oxidized in the presence of the protein, and the
resulting higher oxidation state metal, being a much stronger
Lewis acid, undergoes a hydrolytic polymerization resulting
in the formation of an insoluble metal oxyhydroxide material,
which precipitates from solution inside the cage structure of
ferritin. Mineralization of ferritin using high-oxidation-state
metal ion starting materials has not been successful.
Here we report the photoinduced mineralization of mam-
malian ferritin using high-oxidation-state metal ions as
starting materials. Using this methodology, we have suc-
cessfully mineralized ferritin cages with iron, titanium, and
europim oxyhydroxide nanoparticles in a spatially selective
manner encapsulated within the protein cages. The resulting
materials are uniform and monodispersed and represent a
new synthetic approach to the formation of transition-metal
oxide materials in the nanoscale size regime.
This photochemical reduction closely mimics the photo-
chemical reduction of iron(III) and the subsequent release
of iron(II) observed in marine siderophores that have a citrate
backbone (Figure 1).34,35 Briefly outlined, the ferritin protein
cages were incubated in a 12 mM metal citrate solutions
[metal (M) ) Eu(III), Fe(III), or Ti(IV)], corresponding to
a theoretical loading of 1000 M atoms per ferritin (Supporting
Information), and illuminated with a xenon arc lamp (175
W, Lambda-LS, Sutter Instruments, 320–700 nm), to ap-
proximate solar irradiation, over a 2 h period (for complete
reaction conditions, see the Supporting Information) of the
high-oxidation-state metal precursor is critical because of the
free high-oxidation-state metal ions in solution promotes bulk
Figure 2. Size-exclusion chromatography profile for empty (apo)ferritin
and ferritin containing the europium, titanium, or iron oxyhydroxides
indicating that the mineralization process is occurring on the interior surface
of the protein cage (absorbance monitored at 280 nm).
precipitation. Illumination resulted in the photoreduction of
the high-oxidation-state metal citrate salt to produce transient
low-oxidation-state metal ions that in the presence of air
underwent reoxidation to form a metal oxyhydroxide inside
the protein cages after dissociation of the citrate. In contrast,
control reactions using low-oxidation-state metal ion in the
absence of citrate resulted in bulk precipitation when
illuminated. When this approach was taken using iron(III)
citrate, in air, photolysis resulted in the mineralization of an
Fe(O)OH particle within the protein cage, However, under
N2, no mineralization was observed, and under these condi-
tions, Fe(II) was easily detected by trapping it as the
Fe(phen)32+ complex. This is in agreement with the formation
of Fe(II) by the photolysis reaction. The presence of O2
resulted in the reoxidation to Fe(III), which in the presence
of the apoferritin cage resulted in the spatially selective
mineralization of Fe(O)OH.
A similar approach was utilized for the selective miner-
alization of Eu(O)OH nanoparticles within the ferritin protein
cage. Eu(II) is susceptible to oxidation to Eu(III), and
attempts to use Eu(II) as a precursor for oxidative mineral-
ization of apoferritin resulted in the spontaneous bulk
precipitation of Eu(O)OH. However, when europium(III)
citrate was photolyzed, in the presence of apoferritin, no bulk
precipitation was observed and the reaction remained clear
and homogeneous. In contrast, photolysis of europium(III)
citrate in apoferritin-free control reactions resulted in the
formation of a white precipitate.
The photolysis of titanium(IV) citrate in the presence of
ferritin resulted in the formation of a clear homogeneous
solution. In contrast, when the reaction was carried out in the
absence of protein, a bulk white precipitate was observed. The
reaction appears to proceed by a reduction of the high-oxidation-
state transition metal through excitation of a charge-transfer
transition in the Ti(IV) case. This could be demonstrated in the
case of the Ti reaction when photolysis was performed under
N2, thus preventing the possibility of reoxidation of Ti(III)
formed during the photolysis. Under these conditions, the
characteristic purple color for the d1 Ti(III) species could be
observed within minutes of initiating photolysis.
(25) Meldrum, F. C.; Douglas, T.; Levi, S.; Arosio, P.; Mann, S. J. Inorg.
Biochem. 1995, 58, 59.
(26) Klem, M. T.; Willits, D. A.; Solis, D. J.; Belcher, A.; Young, M.;
Douglas, T. AdV. Funct. Mater. 2005, 15, 1489.
(27) Ueno, T.; Suzuki, M.; Goto, T.; Matsumoto, T.; Nagayama, K.;
Watanabe, Y. Angew. Chem., Int. Ed. 2004, 43, 2527.
(28) Kramer, R. M.; Li, C.; Carter, D. C.; Stone, M. O.; Naik, R. R. J. Am.
Chem. Soc. 2004, 126, 13282.
(29) Ensign, D.; Young, M.; Douglas, T. Inorg. Chem. 2004, 43, 3441.
(30) Galvez, N.; Sanchez, P.; Dominguez-Vera, J. M. Dalton Trans. 2005,
2492.
(31) Wong, K. K. W.; Mann, S. AdV. Mater. 1996, 8, 928.
(32) Yamashita, I.; Hayashi, J.; Hara, M. Chem. Lett. 2004, 33, 1158.
(33) Iwahori, K.; Yoshizawa, K.; Muraoka, M.; Yamashita, I. Inorg. Chem.
2005, 44, 6393.
(34) Barbeau, K. Photochem. Photobiol. 2006, 82, 1505.
(35) Barbeau, K.; Rue, E. L.; Trick, C. G.; Bruland, K. T.; Butler, A.
Limnol. Oceanogr. 2003, 48, 1069.
2238 Inorganic Chemistry, Vol. 47, No. 7, 2008

COMMUNICATION
indicated that the mineralized protein eluted with the same
retention time as the unmineralized apoferritin (Figure 2).
This confirms that the overall particle size of these composite
materials remains unaltered, with the mineral entrapped
within the protein cage interior. No colloidal material is
detected exterior to the protein cage architecture.
The products of the mineralization reactions were further
characterized by transmission electron microscopy (TEM).
Unstained samples showed electron dense cores of a size
consistent with the interior diameter of the protein cage
(Figure 3, left panel). The particle size distributions were
found to be monodispersed with an average diameter of 5.7
( 1 nm. When samples were negatively stained with uranyl
acetate, the intact protein cage was clearly visible as a white
“halo” surrounding the metal oxide cores on the interior
surface (Figure 3, right panel). Electron diffraction was not
observed, suggesting that the nanoparticles generated were
amorphous or poorly crystalline.
This work highlights two important concepts. First, this
photochemical method allowed for the size-constrained
synthesis of previously unattainable oxyhydroxide nanoma-
terials (Eu and Ti) using high-oxidation-state transition-metal
precursors. Second, the synthetic method is general enough
to be applicable to a wide variety of transition-metal/rare-
earth oxyhydroxides, which could be precursors to other
oxide materials.
Figure 3. TEM micrographs of unstained (left) and uranyl acetate stained
(right) photomineralized (A) europium oxyhydroxide, (B) iron oxyhydroxide,
and (C) titanium oxyhydroxide nanoparticles in the protein cage ferritin.
The white “halo” surrounding each particle in the stained images are due
to the protein cage ferritin.
Acknowledgment. This research was supported in part
by grants from the National Science Foundation (Grants
0103509 and NIRT-DMR0210915), the Office of Naval
Research (Grants 19-00-R006 and N00014-03-1-0692), and
the National Institutes of Health (Grant 1R21EB005364).
The products of all of the photolysis reactions were
investigated using dynamic light scattering and found to have
the same size distribution as the original protein cage starting
materials (12 ( 1 nm diameter; Supporting Information).
This indicates the high degree of spatial selectivity with
which the mineralization reaction occurs and suggests that
the inorganic nanomaterials are entrapped within the confines
of the ferritin protein cage. The reaction products were further
analyzed by size-exclusion chromatography. These data
Supporting Information Available: Details on the photoreduc-
tion setup, gel electrophoresis, and dynamic light scattering. This
materialisavailablefreeofchargeviatheInternetathttp://pubs.acs.org.
IC701740Q
Inorganic Chemistry, Vol. 47, No. 7, 2008 2239