Bio-Inspired Growth of Crystals: An Experimental Perspective on Biomineralization

1. Bio-Inspired Growth of Crystals: An Experimental Perspective on Biomineralization Prof. Lara A. Estroff Dept. of Materials Science and Engineering Cornell University lae37@cornell.edu http://laegroup.ccmr.cornell.edu/

2. Biological origin - Coccoliths The White Cliffs of Dover - Limestone (CaCO3)

3. 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 . . . Prof. Heinz A. Lowenstam Geologist, Paleoecologist, Biochemist, and more

4. . . . which could only mean that its teeth were harder than limestone! X-ray diffraction revealed Magnetite (Fe3O4)!

5. Biomineralization and Geology Aragonite • Diversity of Minerals: • Ca5(PO4)3(OH,F) (bone, teeth) • CaCO3 (shells, sea urchins) • SiO2 (plants, sea plankton) • Iron oxides • other carbonates

6. Biomineralization and Geology • Ocean 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.

7. Biomineralization and Geology • Ocean 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)

8. Soluble Proteins Insoluble Matrix • Hydrophilic • Functionality • Nucleation and Growth • Asp/Glu, OPO33-, OSO32- • Hydrophobic • Structural framework • Microenvironment • collagen, chitin, silk fibroin Shell, Teeth, etc How do organisms control crystal growth? Dissolve Mineral Can we experimentally model this complex system both to help us understand the biology and to synthesize new materials with altered properties?

9. Calcite (R-3c) Aragonite (Pcmn) • a = 4.989 Å • c = 17.062 Å a = b = 90° • = 120° Z = 6 • a = 4.961 Å • b = 7.967 Å • c = 5.740 Å • = b = g = 90° Z = 4 Calcite Aragonite Vaterite (hexagonal) 10 µm 5 µm 5 µm Crystal Polymorphs - Calcium Carbonate

10. Crystal Growth as Molecular Recognition additive molecule Weissbuch et. al., Science, 1991

11. -helices - winter flounder -sheet helix - spruce budworm Example: Anti-Freeze Proteins Davies et. al. 2000, Nature

12. Control During Growth: Morphology Mature Sea Urchin Spicule • Diffracts X-Rays as a single crystal • 0.02 wt% protein in mineral • Fractures conchoidally 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% Aizenberg et al., JACS, 1997 Albeck et al., JACS, 1993

13. Regenerating sea urchin spines begin as amorphous CaCO3 Amorphous Precursors to Crystals Politi et al, Science, 2004

14. Control of Nucleation: Orientation and Polymorph Organic Matrix 1 µm Aragonitic nacre layer of a mollusk shell Prismatic calcite layer of a mollusk shell 10 µm 1 µm 5 µm On Biomineralization, 1989, Lowenstam and Weiner

15. New Nacre Model - Colloidal Gel Soluble fraction (10-14 kD, pI <3, Atrina) Silk fibroin (from silk worm cocoon) b-Chitin (squid pen) Levi-Kalisman et al, J. Struct. Bio., 2001. Nudelman, et. al., J. Struct. Biol., 2006 Falini et al., Science, 1996; Levi et al.,Chem. Eur. J., 1998 An in Vitro Model for Nacre

16. New Nacre Model - Colloidal Gel Levi-Kalisman et al, J. Struct. Bio., 2001. Nudelman, et. al., J. Struct. Biol., 2006 An in Vitro Model for Nacre A Hydrogel + A Patterned surface

17. Crystal Growth in Hydrogels • 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. 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?

18. Polysaccharides: Agarose Proteins: Silk Fibroin Estroff and Hamilton, Chem. Rev., 2004 Kim et. al., Biomacromolecules, 2004 Freeze-Dried 1 w/v% agarose gel Types of Hydrogels

19. Self-Assembled Monolayers (SAMs) of alkanethiolson gold • SAMs can control: • Nucleating face • Crystal location • Crystal density Han and Aizenberg, ACIE, 2003 Aizenberg, et al., JACS, 1999; Nature, 1999 Love, Estroff, et. al., Chem. Rev., 2005 How can we control nucleation in the gel?

20. An in vitro Assay to Control Nucleation and Growth Experimental Procedure: Form carboxylate SAMs on gold films. Form a hydrogel (agarose or silk fibroin), with Ca2+, on top of SAM. Expose to atmosphere of CO2 and NH3 to begin the growth of calcium carbonate crystals.

21. An in vitro Assay to Control Nucleation and Growth Experimental Procedure: NH3(g) and CO2(g) (NH4)2CO3 Gel + Ca2+

22. Agarose Gel and Carboxylate-terminated SAM Bulk Agarose Gel (no nucleating surface) Conditions: Agarose gel (2 wt%); CaCl2 (7 mM) Result: Star-shaped calcite and vaterite spherulites Conditions: Agarose gel (2 wt%); CaCl2 (7 mM) Result: truncated rhombohedron of calcite with a (012) orientation Yang et. al., Chem. Commun., 2003 Agarose Hydrogels for Crystal Growth Li and Estroff, J. Am. Chem. Soc., 2007

23. 0 wt% 1 wt% 2 wt% 3 wt% Crystal Shape Changes with [Agarose] Li and Estroff, J. Am. Chem. Soc., 2007

24. Crystal Shape Changes with [Agarose] Li and Estroff, J. Am. Chem. Soc., 2007

25. Pokroy and Aizenberg, CrystEngComm, 2007 Travaille, PhD Thesis, Univ. Nijmegen, 2005 Aspect Ratio and Lattice Mismatch

26. 3 w/v% solution Why does the gel change the aspect ratio? Mass transport: Diffusion vs. convection Presence of organic material: Gel-grown crystals have occluded organic material that may alter the lattice mismatch strain with the SAM.

27. Solution-Grown Crystals Etched Two Days in DI Water Gel-Grown Crystals Etched Two Days in DI Water Is there organic material inside of the crystals?

28. Etch Four Days in DI Water Etch in HCl (0.1 M) 10 min. Ca eV 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?

29. Sea urchin tooth thin section Where are the organic fibers in the crystals? Li and Estroff, CrystEngComm, 2007

30. Mechanisms of Incorporation 2) Growth in Porous Networks Attractive Particle/Crystal Interactions: RT - = D = s p p P c 1 V m p = p ressu r e o n th e c Particle screens mass transport, preventing growth of advancing front; particle is “overtaken” by next layer. At high growth rates, the particle is “pressed” into the crystal by fluid flow, leading to incorporation. l o a ded face o f g r o wi ng cryst al † p = a m b i e n t pr e ssur e l V = m ol ar v o l u me of m so li d phase Chernov, 1984, in Modern Crystallography Khaimov-Mal'kov, Soviet Physics: Crystallography1958

31. 0 % agarose 2 % agarose Increased fracture toughness due to incorporated gel fibers Miki Kunitake Fracture Behavior of Gel-Grown Crystals

32. Fractured Synthetic Calcite Fractured Sea Urchin Spine Fracture Behavior of Gel-Grown Crystals Addadi and Weiner, J. Mater Chem, 1998 Aizenberg and Hendler, J. Mater Chem, 2004

33. Conclusions and Outlook • 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.

34. Acknowledgments Estroff Research Group Gali Baler Jason Dorvee Laura Floyd Ellen Keene Patrick Kiernan Miki Kunitake Hanying Li Debra Lin Mike Lis Vijay Ravichandran Freddy Wang Mike Zettel Funding & Facilities NIH/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)