COMMUNICATION www.rsc.org/materials | Journal of Materials Chemistry

Dow

nloa

ded

on 1

0 Ju

ne 2

012

Publ

ishe

d on

09

Nov

embe

r 20

09 o

n ht

tp://

pubs

.rsc

.org

| do

i:10.

1039

/B91

8620

DView Online / Journal Homepage / Table of Contents for this issue

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: tdouglas@chemistry.montana.edu

† 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.

Dow

nloa

ded

on 1

0 Ju

ne 2

012

Publ

ishe

d on

09

Nov

embe

r 20

09 o

n ht

tp://

pubs

.rsc

.org

| do

i:10.

1039

/B91

8620

D

View Online

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.

Dow

nloa

ded

on 1

0 Ju

ne 2

012

Publ

ishe

d on

09

Nov

embe

r 20

09 o

n ht

tp://

pubs

.rsc

.org

| do

i:10.

1039

/B91

8620

D

View Online

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).

Notes and references

1 A. Fujishima and K. Honda, Nature, 1972, 238, 37.2 R. van de Krol, Y. Q. Liang and J. Schoonman, J. Mater. Chem.,

2008, 18, 2311.3 J. H. Kennedy and K. W. Frese, J. Electrochem. Soc., 1978, 125, 709.4 A. Duret and M. Gratzel, J. Phys. Chem. B, 2005, 109, 17184.5 E. L. Miller, D. Paluselli, B. Marsen and R. E. Rocheleau, Sol. Energy

Mater. Sol. Cells, 2005, 88, 131.6 C. J. Sartoretti, B. D. Alexander, R. Solarska, W. A. Rutkowska,

J. Augustynski and R. Cerny, J. Phys. Chem. B, 2005, 109, 13685.7 A. G. Agrios, I. Cesar, P. Comte, M. K. Nazeeruddin and M. Gratzel,

Chem. Mater., 2006, 18, 5395.8 I. Cesar, A. Kay, J. A. G. Martinez and M. Gratzel, J. Am. Chem.

Soc., 2006, 128, 4582.9 A. Kay, I. Cesar and M. Gratzel, J. Am. Chem. Soc., 2006, 128, 15714.

This journal is ª The Royal Society of Chemistry 2010

10 T. Arai, Y. Konishi, Y. Iwasaki, H. Sugihara and K. Sayama,J. Comb. Chem., 2007, 9, 574.

11 V. M. Aroutiounian, V. M. Arakelyan, G. E. Shahnazaryan,G. M. Stepanyan, E. A. Khachaturyan, H. Wang and J. A. Turner,Sol. Energy, 2006, 80, 1098.

12 U. Bjorksten, J. Moser and M. Gratzel, Chem. Mater., 1994, 6, 858.13 M. M. Khader, G. H. Vurens, I. K. Kim, M. Salmeron and

G. A. Somorjai, J. Am. Chem. Soc., 1987, 109, 3581.14 C. Leygraf, M. Hendewerk and G. A. Somorjai, Proc. Natl. Acad. Sci.

U. S. A., 1982, 79, 5739.15 S. Mohanty and J. Ghose, J. Phys. Chem. Solids, 1992, 53, 81.16 A. B. Murphy, P. R. F. Barnes, L. K. Randeniya, I. C. Plumb,

I. E. Grey, M. D. Horne and J. A. Glasscock, Int. J. HydrogenEnergy, 2006, 31, 1999.

17 P. H. Borse, H. Jun, S. H. Choi, S. J. Hong and J. S. Lee, Appl. Phys.Lett., 2008, 93, 173103.

18 S. W. Lee, C. B. Mao, C. E. Flynn and A. M. Belcher, Science, 2002,296, 892.

19 S. Mann, Biomimetic Materials Chemistry, VCH, New York, 1996.20 S. Mann, D. D. Archibald, J. M. Didymus, T. Douglas,

B. R. Heywood, F. C. Meldrum and N. J. Reeves, Science, 1993,261, 1286.

21 R. R. Naik, S. J. Stringer, G. Agarwal, S. E. Jones and M. O. Stone,Nat. Mater., 2002, 1, 169.

22 M. Uchida, M. T. Klem, M. Allen, P. Suci, M. Flenniken, E. Gillitzer,Z. Varpness, L. O. Liepold, M. Young and T. Douglas, Adv. Mater.,2007, 19, 1025.

23 M. Allen, D. Willits, J. Mosolf, M. Young and T. Douglas, Adv.Mater., 2002, 14, 1562.

24 A. Sidorenko, T. Krupenkin, A. Taylor, P. Fratzl and J. Aizenberg,Science, 2007, 315, 487.

25 W. Shenton, T. Douglas, M. Young, G. Stubbs and S. Mann, Adv.Mater., 1999, 11, 253.

26 M. Allen, D. Willits, M. Young and T. Douglas, Inorg. Chem., 2003,42, 6300.

27 A. S. Blum, C. M. Soto, C. D. Wilson, T. L. Brower, S. K. Pollack,T. L. Schull, A. Chatterji, T. W. Lin, J. E. Johnson, C. Amsinck,P. Franzon, R. Shashidhar and B. R. Ratna, Small, 2005, 1, 702.

28 T. Douglas, D. P. E. Dickson, S. Betteridge, J. Charnock,C. D. Garner and S. Mann, Science, 1995, 269, 54.

29 T. Douglas and M. Young, Nature, 1998, 393, 152.30 S. Mann and G. A. Ozin, Nature, 1996, 382, 313.31 F. C. Meldrum, B. R. Heywood and S. Mann, Science, 1992, 257,

522.32 B. Wiedenheft, J. Mosolf, D. Willits, M. Yeager, K. A. Dryden,

M. Young and T. Douglas, Proc. Natl. Acad. Sci. U. S. A., 2005,102, 10551.

33 B. Wiedenheft, M. L. Flenniken, M. A. Allen, M. Young andT. Douglas, Soft Matter, 2007, 3, 1091.

34 M. J. Parker, M. A. Allen, B. Ramsay, M. T. Klem, M. Young andT. Douglas, Chem. Mater., 2008, 20, 1541.

35 M. T. Klem, D. Willits, D. J. Solis, A. M. Belcher, M. Young andT. Douglas, Adv. Funct. Mater., 2005, 15, 1489.

36 B. Ramsay, B. Wiedenheft, M. Allen, G. H. Gauss, C. M. Lawrence,M. Young and T. Douglas, J. Inorg. Biochem., 2006, 100, 1061.

37 J. Tatur, P. L. Hagedoorn, M. L. Overeijnder and W. R. Hagen,Extremophiles, 2006, 10, 139.

38 I. G. Macara, P. M. Harrison and T. G. Hoy, Biochem. J., 1972, 126,151.

39 Z. Pu, M. Cao, J. Yang, K. Huang and C. Hu, Nanotechnology, 2006,17, 799.

40 F. M. Michel, L. Ehm, S. M. Antao, P. L. Lee, P. J. Chupas, G. Liu,D. R. Strongin, M. A. A. Schoonen, B. L. Phillips and J. B. Parise,Science, 2007, 316, 1726.

41 H. Park, W. Choi and M. R. Hoffmann, J. Mater. Chem., 2008, 18,2379.

42 H. W. Park, J. S. Lee and W. Y. Choi, Catal. Today, 2006, 111, 259.43 J. D. Desai, H. M. Pathan, S. K. Min, K. D. Jung and O. S. Joo, Appl.

Surf. Sci., 2005, 252, 1870.

J. Mater. Chem., 2010, 20, 65–67 | 67