COMMUNICATION www.rsc.org/materials | Journal of Materials Chemistry
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Biomimetic synthesis of photoactive a-Fe2O3 templated by thehyperthermophilic ferritin from Pyrococus furiosus†
Michael T. Klem,a Mark Youngbc and Trevor Douglas*c
Received 8th September 2009, Accepted 27th October 2009
First published as an Advance Article on the web 9th November 2009
DOI: 10.1039/b918620d
Utilizing a biomimetic approach toward materials synthesis
a ferritin protein cage, from the hyperthermophilic archeaon Pyro-
coccus furiosus, was utilized to first tempate the formation of
a largely amorphous ferrihydrite iron oxide and then bring about its
transformation to hematite (a-Fe2O3). This was achieved under
boiling aqueous conditions by refluxing the ferritin protein cage/
ferrihydrite composite. The resultant material showed diffraction
indicative of hematite, a visible band gap semiconductor, and
photocurrents were measured under visible illumination. The resul-
tant protein/mineral composite was also studied via dynamic light
scattering, transmission electron microscopy, and size exclusion
chromatography.
In an effort to find alternatives to nonrenewable fossil fuels,
researchers have focused on using sunlight to evolve hydrogen as
a clean and renewable energy source. Fujishima and Honda using
a TiO2 photoanode experimentally achieved direct light-induced
water splitting.1 Hematite (a-Fe2O3) has also attracted interest as
a photoanode because of its nearly ideal band gap, visible light
absorption, nontoxicity, and excellent chemical stability under
appropriate conditions.2 One of the difficulties in using hematite as
a photoanode is its extremely short hole diffusion length of approx-
imately 5 nm.3 This short diffusion length is the prevailing cause of
the low efficiencies observed with iron-oxide photoanodes due to
electron hole recombination.
It has been hypothesized that creation of nanoparticles of a-Fe2O3
with sizes commensurate with the hole diffusion length would exhibit
improved efficiencies for photoanodic oxidation because of the
hindrance of charge transport inside the nanoparticles. Currently,
high temperature vapor phase methods have been employed in the
fabrication of iron oxide photoanodes, but these methods have had
limited success in producing crystalline iron oxides with particle sizes
in the region of 5 nm.3–6 The use of nanostructured films7–9 and
heteroatom dopants4,10–16 have been explored as pathways to
aDepartment of Chemistry & Geochemistry, Montana Tech of theUniversity of Montana, Butte, MT, USAbDepartment of Plant Sciences, Center for BioInspired Nanomaterials,NASA Astrobiology Biogeocatalysis Center Montana State University,Bozeman, MT, USAcDepartment of Chemistry and Biochemistry, Center for BioInspiredNanomaterials, NASA Astrobiology Biogeocatalysis Center MontanaState University, Bozeman, MT, USA. E-mail: [email protected]
† Electronic supplementary information (ESI) available: Pf_Fnpurification from E.coli and characterization details. See DOI:10.1039/b918620d
This journal is ª The Royal Society of Chemistry 2010
improved performance. The incorporation of nanoparticles in thin
films have also been investigated.17
Biomimetic approaches toward materials chemistry have provided
new avenues for the synthesis and assembly of nanomaterials.18–22
Bio-inspired approaches in material synthesis have utilized evolved
molecular interactions,23,24 macromolecular templates,25 and well
defined protein cage architectures26–31 to exert synthetic control over
crystal morphology, phase, and orientation. While biological
macromolecules have been utilized in materials synthesis their use has
often been limited by their relatively low thermal stability compared
with other synthetic methods. Isolation of protein templates from
thermophilic and hyperthermophilic organisms is one approach
which might ease this limitation.32,33 The isolation and synthetic
utilization of a number of thermally stable proteins such as ferritins,34
heat shock proteins,35 and DNA-binding proteins from starved
cells32,36 from hyperthermophilic archaea have significantly expanded
the temperature range for in vitro biomimetic synthesis.
Recently, a hyperthermophilic ferritin was isolated from the
archaeon Pyrococcus furiosus, a marine anaerobe that lives in high
temperature environments where temperatures can reach 120 �C.37
This protein cage has been cloned from its original host, and over-
expressed in Escherichia coli.37 The recombinant Pyrococcus ferritin
(Pf_Fn) was subjected to reaction temperatures of 120 �C and was
shown to retain the iron sequestering behavior for over 30 min at
these extreme temperatures.37 The ability of this protein to template
magnetic iron oxides (i.e. Fe3O4) has also been demonstrated at
elevated temperatures with magnetic properties different from
syntheses using mesophillic ferritin as a template.34
Ferrihydrite was initially synthesized inside Pf_Fn with a chemical
loading of 1000 Fe atoms/cage using previously established
methods.38 Isolated Apo-Pf_Fn protein cages were diluted to 0.3 mg
mL�1 in 100 mM MES (pH ¼ 6.5) and 50 mM NaCl. Dearated
solutions of (NH4)2FeSO4$6H2O were added to the Pf_Fn for a total
chemical loading of 1000 Fe/cage over a period of 24 h. The resulting
solution was allowed to air oxidize to form 7.0 nm ferrihydrite
particles. The Pf_Fn/ferrihydrite composite was dialyzed into 50 mM
HEPES (pH ¼ 7.0) and 100 mM NaCl. The solution was then
refluxed at 97 �C in the presence of trace amounts of sodium oxalate
(<1 mM) for 1–5 days.
Using a biomimetic approach we have developed a synthetic
technique that involves the conversion of a relatively amorphous
hexagonal iron oxide (ferrihydrite) to ordered a-Fe2O3 within the
Pf_Fn protein cage. Ferrihydrite was converted to hematite using
processes previously described as a combination of catalytic disso-
lution/reprecipitation and topotactic solid-state transformation inside
the Pf_Fn protein cage.39 The pre- and post- reaction products were
characterized by TEM (Fig. 1). Unstained samples showed electron-
dense cores whose sizes (6.9 � 1.1 nm) were commensurate with the
J. Mater. Chem., 2010, 20, 65–67 | 65
Fig. 1 Transmission electron micrographs of (A) unstained, (B) stained
refluxed Pf_Fn, and (C) unstained ferrihydrite in Pf_Fn. The dark elec-
tron dense cores in (A) have an electron diffraction pattern (insert)
consistent with a-Fe2O3 while no diffraction was observed in (C)
consistent with disordered ferrihydrite.
Fig. 2 Size exclusion chromatograph of empty (——), FeOOH containing
(/), and a-Fe2O3 (----) Pf_Fn protein cages.
Fig. 3 Photocurrent generated upon exposure of ferrihydrite in Pf_Fn
(----), Methyl viologen (——), and hematite Pf_Fn/Methyl viologen (--.-) to
a high intensity LED light.
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interior dimensions of the protein cage. When samples were nega-
tively stained with uranyl acetate, the intact protein cages were clearly
visible as a white ‘‘halo’’ surrounding the metal oxide cores on the
interior surface. Electron diffraction was not observed in the ferri-
hydrite samples consistent with the relatively amorphous nature of
ferrihydrite.40 Electron diffraction, with d-spacings corresponding to
a-Fe2O3, was observed in the refluxed samples (ESI†). Lower
temperature phase transformations (i.e. 65 �C) using Pf_Fn cages
were unsuccessful at generating hematite nanoparticles from the
ferrihydrite precursor. Control reactions using horse spleen ferritin
were also unsuccessful at generating hematite nanoparticles from
a ferrihydrite precursor due to precipitation of the protein at the high
temperatures required.
The products were further characterized using dynamic light
scattering and found to have the same size distribution as the original
protein cage starting materials, 12.9 � 0.5 nm vs. 13.0 � 0.2 nm for
unheated and heated, respectively (ESI†). This indicates the high
degree of heat stability and spatial selectivity the protein cage exerts
on the iron oxides. No colloidal material was detected exterior to the
Pf_Fn protein cages. The protein absorbance at 280 nm was used to
monitor elution of the composite protein/mineral by size exclusion
chromatography (Fig. 2). The mineralized product had an elution
profile with a retention time identical to that of empty Pf_Fn protein.
This co-elution behavior points toward the composite nature of the
material and suggests that the protein cage and mineral are intimately
associated. Size exclusion chromatography is relatively sensitive to
small changes in particle size, and thus these data suggest that the size
of the protein cage has not been significantly perturbed by the reac-
tion. It is also indicative of the mineral being encapsulated within the
protein cage architecture demonstrating the spatial selectivity of the
reaction.
Photocurrents generated from the a-Fe2O3/Pf_Fn composite were
collected at an ITO working electrode immersed in a suspension of
a-Fe2O3/Pf_Fn (0.3 mg mL�1 a-Fe2O3/Pf_Fn, ethanol) using methyl
66 | J. Mater. Chem., 2010, 20, 65–67
viologen (MV2+, 2 mM) as an electron mediator.41,42 The photocur-
rents generated under illumination from a high intensity 420 nm LED
are shown (Fig. 3). Conversely, control reactions involving the empty
Pf_Fn cage and MV2+ exhibited photocurrents on the order of
0.0–0.5 mA. Photo-reactions performed with the a-Fe2O3/Pf_Fn
composite gave rise to the photocurrents on the order of 5–37 mA
cm�2 depending on concentration (Fig. 4). The maximum in the
current density is most likely to be due to incomplete conversion of
the ferrihydrite precursor to hematite. Further studies also indicate
that the transformation is complete within 60 min of reaction. No
further gain in photocurrent was observed after 60 min (Fig. 5).
Despite the favorable band gap for a-Fe2O3, the photocurrents
observed are lower than those observed for sprayed a-Fe2O3 films.43
It is hypothesized that interactions between the protein cage and the
photoexcited electron allow for electron/hole recombination to occur
before the MV2+ can be reduced. This is most likely to be due to the
mass transport limitation of MV into and out of the protein cage.
This work illustrates two important concepts. First, a ferritin
protein cage isolated from a hyperthermophilic archea can be effec-
tively used as a size constrained reaction vessel for the conversion of
ferrihydrite to a-Fe2O3 under boiling aqueous conditions. Further-
more, the inherent temperature stability of this ferritin is a necessary
requirement for a successful transformation. Second, the resultant
hematite/protein composite acts as a visible band gap semiconductor
This journal is ª The Royal Society of Chemistry 2010
Fig. 4 Photocurrents generated as a function of protein concentration.
Fig. 5 Photocurrents generated at 60 min vs. 24 h. The strong similar-
ities in the currents observed suggest that the conversion of ferrihydrite to
hematite is complete in one hour.
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for the photoreduction of methyl viologen using ethanol as a sacrifi-
cial reductant.
This research was supported in part by grants from the National
Science Foundation (CBET-0709358), and the Department of
Energy (DE-FG02-07ER46477).
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