A Bioengineering Approach to Environmental Remediation

Daniel Strongin1, Trevor Douglas2, and Martin A. Schoonen3

(1) Department of Chemistry, Temple University, 1901 N. 13th St., Philadelphia, PA (2) Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT

(3) Department of Geosciences, SUNY-Stony Brook, Stony Brook, NY

R829601

ACS-PRF

Acknowledgments

Hazel-Ann Hosein - Temple Univ.Mark Allen - Montana State Univ.Dan Ensign - Montana State Univ.

Horse Spleen Ferritin (HSF)

Listeria Innocua Ferritin-like Protein (LFLP)

• 24 polypeptide subunits

• Spherical protein cage (120 Å dia.)

• Cavity (80 Å dia.)

• Accommodates up to 4500 Fe atoms

Stores Fe as hydrated Fe2O3 (rust)

• 12 polypeptide subunits

• Spherical protein cage (90 Å dia.)

• Cavity (56Å dia.)

• Accommodates up to 500 Fe atoms

12 distinct sites10 Glu residues (at each site)

Glutamate (COO—)

N

N

N

N

N

N

RuII

HN

OS

Ru(phen)(bpy)22+ covalently linked to HSP G41C = photosensitizer

Small Heat Shock Protein Cage

From the thermophile

Cloned Mutant used

24 subunit cage with large 3nm

Diameter pores

Ferritin as a (photo)catalyst

Ferritin as a Template forThe growth of oxide and Metallic nanoparticles

Functionalization of the ferritin shell

Goals of EPA - Funded Research

2Fe2+ + Prot Prot-[Fe2]O2

Prot-[Fe2–O2]

Prot-[Fe-O-Fe]

H2O2

H2O, H+

2Fe2+ + O2

Fe(O)OH(s)

H2O2

2Fe(O)OH + 4 H+ Ferroxidase catalysed (important early on in the mineralization process )

4Fe2+ + O2 4Fe(O)OH + 2H2O + 8 H +Mineral catalysed (autocatalytic Fe(II) oxidation and hydrolysis

Fe Fe

O––O

OC

OO

CO

Hwang, J., Krebs, C., Huynh, B. H., Edmondson, D. E., Theil, E. C. & Penner-Hahn, J. E. (2000). Science 287, 122

h

reductionby e-

oxidationby h+

e-

h+

A

A-

D

D+

oxidecore

Shell

Channel

Ferritin as a Photocatalyst

TiO2 vs. Ferric oxides

TiO2

Bandgap=3.2 eV uv lamps needed to

maximize photochemistry

very high stability

Ferric oxides (e.g., FeOOH

Bandgap=2.2 - 2.8eV can utilize a significant

part of solar spectrum low stability -

photocorrosion

Xenon vs. Solar spectrum

Xenon: The Full Spectrum vs. Deuterium Plus Tungsten by Robert A. Capobianco, http://opto.perkinelmer.com/library/papers/tp9.asp

TiO2

FeOOH

Experimental

Crlamp

-0.6

-0.5

-0.4

-0.3

-0.2

-0.1

0.0D

ecr

ea

se o

f th

e A

bso

rba

nce

at

37

2n

m

20151050

Time, min

a

b

c

The decrease of the absorbance at 372 nm. The final concentration of the species: Na2Cr2O7: 5x10-6M; tartrate: 3x10-2M; Ferritins: 0.25mg/ml; Buffer: 0.1M. Tris, pH8.5.a, CoOOH-Fn; b, MnOOH-Fn; c, FeOOH-Fn.

Cr(VI) Cr(III)

lightCr2O7

2- + 14H+ + 6e- 2Cr3+ 7H2O

Can we tailor a nanoparticle system having a bandgap In the visible with high stability for application in environmental remediation?

Stability of catalytic ferritin particles

Time (min)0 50 100 150 200 250 300

ln[C

r(V

I)/C

r(V

I)o

]

-3.5

-3.0

-2.5

-2.0

-1.5

-1.0

-0.5

0.0

0.5

5:110:115:120:1

Cr:Fe

Reductant – tartrate (3.2x10-3 M)

Cr2O72- (4.0x10-4 M)

pH 7.5, tris buffer

Ferritin catalyzed reduction

Cr(III)

h

Cr(VI)

e-

Cr(III)

reductant

e-

h+

Photocorrosion and aggregation of protein free FeOOH

h

Fe(II)O2 , H+

Insoluble precipitate

h

Fe(II)Fe(III)

O2

Ferritin system

Ferritin as a Template forThe growth of oxide and Metallic nanoparticles

Apo-Ferritin

120

80

Ferritin Fe(O)OH

Co(O)OH Mn (O)OH Fe(O)OH

Fe Co

Reduction

Loading ofmetal controlsultimate nanoparticlesize

UV-Ozone treatment

Reduction

General synthetic scheme for Fe metal production

The PSD-UV uses high intensity UV radiation to vaporize and remove the protein portion

The high pressure cell coupled to UHV chamber where reduction of metal oxide to metal occurs and accompanying transfer apparatus.

Heights (nm

0.5 1 1.5 2 2.5 3 3.5

Fre

qu

en

cy

0

5

10

15

20

FeOOH nanoparticles prepared by UV-ozone treatment of 100 Fe loaded ferritin for 60 mins at 100oC under oxygen (<5psi)

ISOLATED NANOPARTICLES

Average Height ONLY 2.5 nm!

Acoustic AC mode AFM Characterization of FeOOH nanoparticles

Relative height distribution of particles

AC Mode AFM Images: Apoferritin + FeFn mixturesApoferritin + 500 FeFn mixture Apoferritin + 2000 FeFn mixture

TM-AFM Characterizationof Iron nanoparticles

• ISOLATED NANOPARTICLES• Peak-to-valley height differences for the large features in the cross-section are in the 4-6 nm range.

• Full range of height values = 8.0 nm

• RMS roughness = 1.47 nmFe nanoparticles prepared by heating FeOOHnanoparticles in H2.

Height mode; 286 x 286 nm

TM-AFM Characterization

of Co3O4 nanoparticles

Height mode; 235 x 235 nm

Co3O4 nanoparticles on SiO2 prepared by UV-ozone treatment of LFLP for 1 hr at 100oC under oxygen (<5psi)

Narrow Distribution of particle sizes: 2.5-3.0 nm

RMS roughness = 1.07 nm

citrate

Cu2+ Cuº

1.5

1.0

0.5Ab

sorb

an

ce

900800700600500Wavelength, nm

D. Ensign, M. Young, T. Douglas Inorg. Chem (2004)

Photocatalytic Synthesis of Cu nanoparticles

QuickTime™ and aGIF decompressor

are needed to see this picture.

Cu2+

e-

h+

h

Cu0

Fe(O)OH

1) 2)

15

10

5

0

nu

mb

er

of

part

icle

s

20151050diameter, nm

500 Cu:ferritin

6

5

4

3

2

1

0n

um

ber

of

part

icle

s

6050403020100diameter, nm

2000 Cu:ferritin

500 Cu/Fn 2000 Cu/Fn

9.7 ± 3 nm 31 ± 8 nm

QuickTime™ and aGIF decompressor

are needed to see this picture.

As(III) addition

2.5 m

200 nm

200 nm

Wide scan

Insolubleprecipitate

Sequestration ofAqueous As(III) byFeOOH nanoparticles

FeOOH NanoparticlesSupported on Si wafer

Atomic Force Microscopy

No nanoparticlesNo precipitate

Visible Light-Induced Production of Singlet OxygenGround triplet state 3g

First excited singlet state 1g

Second excited singlet state 1g

relax-

ation

2s

* 2s 2p

* 2p* 2p 2p

3g

1 g

1g

h

96.5 kJ/mol

h

254 kJ/mol

1GS

1MLCT3MLCT

Eh

3O2

1O2

photosensitizer

Why?• Delivery of photosensitizers to cells

• Illumination produces 1O2

• 1O2 is about 1 V more oxidizing than 3O2

Functionalization of the ferritin

Visible Light-Induced Production of Singlet Oxygen

N

N

N

N

N

N

RuII

HN

OS

Ru(phen)(bpy)22+ covalently linked to HSP G41C = photosensitizer

h

3O2

1O2

Excitation of the Ru(II)-HSP complex increases the rateOf singlet oxygen production by a factor of 50 comparedTo the Ru(II) complex alone!

SUMMARYFerritin facilitates

Reduction of Cr(VI) to Cr(III)

Ferritin as a Template for the growth of oxide and metallic nanoparticlesFe and Co oxides / Fe, Co, and Cu metals

Functionalization of the ferritin-like protein shell to form photosensitizer

Acknowledgments

Hazel-Ann Hosein - Temple Univ.Mark Allen - Montana State Univ.Dan Esign - Montana State Univ.