Bio-Inspired Growth of Crystals:An Experimental Perspective on

Biomineralization

Prof. Lara A. EstroffDept. of Materials Science and Engineering

Cornell Universitylae37@cornell.edu

http://laegroup.ccmr.cornell.edu/

The White Cliffs of Dover - Limestone (CaCO3)

Biological origin - Coccoliths

Prof. Heinz A. LowenstamGeologist, Paleoecologist, Biochemist, and more

A father of the field of Biomineralization

On a trip to Bermuda, Prof. Lowenstam noticed this creature, a chiton, making “chevron” marks on a limestone surface . . .

. . . which could only mean that its teeth were harder than limestone!

X-ray diffraction revealedMagnetite (Fe3O4)!

Biomineralization and Geology

Diversity of Minerals:

- Ca5(PO4)3(OH,F) (bone, teeth) - CaCO3 (shells, sea urchins)- SiO2 (plants, sea plankton)- Iron oxides- other carbonates

Aragonite

Biomineralization and GeologyOcean Carbon Cycle:

• Cocoliths, foraminiferas, and coral are key players.• Balance between photosynthesis and calcification. • pH sensitivity of organisms with calcified skeletons.• Solubilities of biogenic CaCO3 is different from geological minerals and varies species to species.

Biomineralization and GeologyOcean Carbon Cycle:

• Cocoliths, foraminiferas, and coral are key players.• Balance between photosynthesis and calcification. • pH sensitivity of organisms with calcified skeletons.• Solubilities of biogenic CaCO3 is different from geological minerals and varies species to species.

M. Fine et al., Science 315, 1811 (2007)

- Hydrophobic- Structural framework

- Microenvironment- collagen, chitin, silk fibroin

Insoluble Matrix- Hydrophilic - Functionality

- Nucleation and Growth- Asp/Glu, OPO3

3-, OSO32-

Soluble Proteins

Shell, Teeth, etc

DissolveMineral

How do organisms control crystal growth?

Can we experimentally model this complex system both to help us understand the biology and to synthesize new materials with altered properties?

Crystal Polymorphs - Calcium CarbonateCalcite (R-3c) Aragonite (Pcmn)

Calcite Aragonite Vaterite (hexagonal)

10 µm 5 µm 5 µm

a = 4.989 Åc = 17.062 Å

90°= 120°

Z = 6

a = 4.961 Åb = 7.967 Åc = 5.740 Å

90°Z = 4

Crystal Growth as Molecular Recognition

Weissbuch et. al., Science, 1991

additive molecule

Example: Anti-Freeze Proteins-helices - winter flounder -sheet helix - spruce budworm

Davies et. al. 2000, Nature

Control During Growth: Morphology

Amino Acid Composition (>3%):AsX 15.5% Ala 8.0%GlX 12.7% Val 3.9%Ser 4.4% Leu 3.5%Thr 6.5% Pro 10.1%Gly 19.4% Arg 5.9%

Mature Sea Urchin Spicule

Aizenberg et al., JACS, 1997Albeck et al., JACS, 1993

• Diffracts X-Rays as a single crystal• 0.02 wt% protein in mineral

• Fractures conchoidally

Amorphous Precursors to Crystals

Politi et al, Science, 2004

Regenerating sea urchin spines begin as amorphous CaCO3

On Biomineralization, 1989, Lowenstam and Weiner

Control of Nucleation: Orientation and Polymorph

Aragonitic nacre layer of a mollusk shell

Prismatic calcite layer of a mollusk shell

Organic Matrix

1 µm 1 µm

10 µm

5 µm

An in Vitro Model for Nacre

Soluble fraction (10-14 kD, pI <3, Atrina)

Silk fibroin (from silk worm cocoon)-Chitin (squid pen)

Falini et al., Science, 1996; Levi et al., Chem. Eur. J., 1998Levi-Kalisman et al, J. Struct. Bio., 2001.Nudelman, et. al., J. Struct. Biol., 2006

New Nacre Model - Colloidal Gel

An in Vitro Model for Nacre

Levi-Kalisman et al, J. Struct. Bio., 2001.Nudelman, et. al., J. Struct. Biol., 2006

New Nacre Model - Colloidal Gel

A Hydrogel+

A Patterned surface

The chemical environment of nucleation is different in a gel than in a saturated solution:

• Diffusion dominates (convection is suppressed). • High supersaturations

• Hydrophobic gels can “structure” water and proteins.

Crystal Growth in Hydrogels

Questions• Why do organisms use hydrogels to control crystal

growth?• What rules govern the growth mechanisms of crystals in

different types of hydrogels?• Can we apply crystal growth in gels to non-biological

materials (e.g., organic crystals, oxides) to obtain crystals with defined morphologies or mechanical properties?

Types of HydrogelsProteins: Silk Fibroin

Estroff and Hamilton, Chem. Rev., 2004Kim et. al., Biomacromolecules, 2004

OO

OO

OH

OH

OO

OO

OH

OH

O

HO

HOH2C O

HOHOH2C O

n

Polysaccharides: Agarose

Freeze-Dried 1 w/v% agarose gel

How can we control nucleation in the gel?

Han and Aizenberg, ACIE, 2003Aizenberg, et al., JACS, 1999; Nature, 1999

Self-Assembled Monolayers (SAMs) of alkanethiolson gold

Love, Estroff, et. al., Chem. Rev., 2005

SAMs can control:- Nucleating face- Crystal location- Crystal density

An in vitro Assay to Control Nucleation and Growth

1) Form carboxylate SAMs on gold films.2) Form a hydrogel (agarose or silk fibroin), with Ca2+, on

top of SAM.3) Expose to atmosphere of CO2 and NH3 to begin the

growth of calcium carbonate crystals.

Experimental Procedure:

An in vitro Assay to Control Nucleation and Growth

(NH4)2CO3 Gel + Ca2+

NH3(g) and CO2(g)

Experimental Procedure:

Agarose Hydrogels for Crystal Growth

Yang et. al., Chem. Commun., 2003

Conditions: Agarose gel (2 wt%); CaCl2 (7 mM)

Result: Star-shaped calcite and vaterite spherulites

Bulk Agarose Gel (no nucleating surface)

Li and Estroff, J. Am. Chem. Soc., 2007

Agarose Gel and Carboxylate-terminated SAM

Conditions: Agarose gel (2 wt%); CaCl2 (7 mM)

Result: truncated rhombohedron of calcite with a (012) orientation

Crystal Shape Changes with [Agarose]

0 wt% 1 wt%

2 wt% 3 wt%

Li and Estroff, J. Am. Chem. Soc., 2007

Crystal Shape Changes with [Agarose]

Li and Estroff, J. Am. Chem. Soc., 2007

Aspect Ratio and Lattice Mismatch

Pokroy and Aizenberg, CrystEngComm, 2007Travaille, PhD Thesis, Univ. Nijmegen, 2005

Mass transport: Diffusion vs. convectionPresence of organic material: Gel-grown crystals have occluded organic material that may alter the lattice mismatch strain with the SAM.

Why does the gel change the aspect ratio?3 w/v% solution

Is there organic material inside of the crystals?

Gel-Grown Crystals Etched Two Days in DI Water

Solution-Grown Crystals Etched Two Days in DI Water

Etch Four Days in DI WatereV

Ca

Etch in HCl (0.1 M) 10 min.

Continued Etching - Agarose “Crystal Ghosts”

Questions to Answer:

• Why does the crystal grow around the impurity rather than exclude it?• Are the crystals single crystals or “mesocrystals”?• How does the incorporated material alter the mechanical properties of the crystals?

Where are the organic fibers in the crystals?

Sea urchin tooth thin section

Li and Estroff, CrystEngComm, 2007

Mechanisms of Incorporation

Chernov, 1984, in Modern Crystallography

1) Attractive Particle/Crystal Interactions:a) Particle screens mass transport,

preventing growth of advancing front; particle is “overtaken” by next layer.

b) At high growth rates, the particle is “pressed” into the crystal by fluid flow, leading to incorporation.

Khaimov-Mal'kov, Soviet Physics: Crystallography 1958

†

pc p1

P RTVm

pc = pressure on theloaded face of growing

crystalpl = ambient pressureVm = molar volume of

solid phase

2) Growth in Porous Networks

Miki Kunitake

Increased fracture toughness due to incorporated gel fibers

0 % agarose 2 % agarose

Fracture Behavior of Gel-Grown Crystals

Fractured Sea Urchin SpineFractured Synthetic Calcite

Addadi and Weiner, J. Mater Chem, 1998Aizenberg and Hendler, J. Mater Chem, 2004

Fracture Behavior of Gel-Grown Crystals

• Biomineralization and geology have a lot to offer each other.

• The use of synthetic models (e.g., the SAM/Gel matrix) provides insight into the organic-inorganic interface in biominerals.

• Computation can help us to model this molecular scale recognition and, hopefully, design better matrices.

• A fundamental understanding of biomineral growth and dissolution has implications in the global carbon and silicate cycles.

Conclusions and Outlook

Acknowledgments

Funding & FacilitiesNIH/NIDCR (R21)

CCMR Seed Grant (NSF-DMR MRSEC)

CCMR REU program

J.D. Watson Young Investigator Award (NYSTAR)

ACS-PRF

Weill-Ithaca Seed Funding

Engineering Learning Initiative (Cornell)

Gali Baler

Jason Dorvee

Laura Floyd

Ellen Keene

Patrick Kiernan

Miki Kunitake

Hanying Li

Debra Lin

Mike Lis

Vijay Ravichandran

Freddy Wang

Mike Zettel

Estroff Research Group