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Calcium carbonate nucleation driven by ion binding in a biomimetic matrix revealed by in situ electron microscopy

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The characteristic shapes, structures and properties of biominerals arise from their interplay with a macromolecular matrix1,2. The developing mineral interacts with acidic macromolecules, which are either dissolved in the crystallization medium or associated with insoluble matrix polymers3, that affect growth habits and phase selection or completely inhibit precipitation in solution4,5,6. Yet little is known about the role of matrix-immobilized acidic macromolecules in directing mineralization. Here, by using in situ liquid-phase electron microscopy to visualize the nucleation and growth of CaCO3 in a matrix of polystyrene sulphonate (PSS), we show that the binding of calcium ions to form Ca–PSS globules is a key step in the formation of metastable amorphous calcium carbonate (ACC), an important precursor phase in many biomineralization systems7. Our findings demonstrate that ion binding can play a significant role in directing nucleation, independently of any control over the free-energy barrier to nucleation.

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Figure 1: Rapid vaterite formation in the absence of PSS.
Figure 2: Analysis of the formation, morphology and chemistry of as-prepared Ca–PSS globules.
Figure 3: Nucleation of amorphous calcium carbonate from Ca–PSS globules imaged in LP-TEM.
Figure 4: Mechanism of CaCO3 mineral formation in the biomimetic matrix.

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  1. Lowenstam, H. A. & Weiner, S. On Biomineralization (Oxford Univ. Press, 1989).

    Google Scholar 

  2. Mann, S. Biomineralization, Principles and Concepts in Bioinorganic Materials Chemistry (Oxford Univ. Press, 2001).

    Google Scholar 

  3. Special issue on Biomineralization. Chem. Rev. 108, 4329–4978 (2008).

    Article  CAS  Google Scholar 

  4. Sommerdijk, N. & de With, G. Biomimetic CaCO3 mineralization using designer molecules and interfaces. Chem. Rev. 108, 4499–4550 (2008).

    Article  CAS  Google Scholar 

  5. Meldrum, F. C. & Colfen, H. Controlling mineral morphologies and structures in biological and synthetic systems. Chem. Rev. 108, 4332–4432 (2008).

    Article  CAS  Google Scholar 

  6. Gower, L. B. Biomimetic model systems for investigating the amorphous precursor pathway and its role in biomineralization. Chem. Rev. 108, 4551–4627 (2008).

    Article  CAS  Google Scholar 

  7. Addadi, L., Raz, S. & Weiner, S. Taking advantage of disorder: Amorphous calcium carbonate and its roles in biomineralization. Adv. Mater. 15, 959–970 (2003).

    Article  CAS  Google Scholar 

  8. Nudelman, F., Chen, H. H., Goldberg, H. A., Weiner, S. & Addadi, L. Spiers Memorial Lecture: Lessons from biomineralization: Comparing the growth strategies of mollusc shell prismatic and nacreous layers in Atrina rigida. Faraday Discuss. 136, 9–25 (2007).

    Article  CAS  Google Scholar 

  9. Young, J. R., Davis, S. A., Bown, P. R. & Mann, S. Coccolith ultrastructure and biomineralisation. J. Struct. Biol. 126, 195–215 (1999).

    Article  CAS  Google Scholar 

  10. Olszta, M. J. et al. Bone structure and formation: A new perspective. Mater. Sci. Eng. R-Rep. 58, 77–116 (2007).

    Article  Google Scholar 

  11. Addadi, L., Moradian, J., Shay, E., Maroudas, N. G. & Weiner, S. A chemical-model for the cooperation of sulfates and carboxylates in calcite crystal nucleation—relevance to biomineralization. Proc. Natl Acad. Sci. USA 84, 2732–2736 (1987).

    Article  CAS  Google Scholar 

  12. Marsh, M. E. Polyanion-mediated mineralization—assembly and reorganization of acidic polysaccharides in the Golgi system of a coccolithophorid alga during mineral deposition. Protoplasma 177, 108–122 (1994).

    Article  CAS  Google Scholar 

  13. Nudelman, F., Gotliv, B. A., Addadi, L. & Weiner, S. Mollusk shell formation: Mapping the distribution of organic matrix components underlying a single aragonitic tablet in nacre. J. Struct. Biol. 153, 176–187 (2006).

    Article  CAS  Google Scholar 

  14. Marsh, M. E., Ridall, A. L., Azadi, P. & Duke, P. J. Galacturonomannan and Golgi-derived membrane linked to growth and shaping of biogenic calcite. J. Struct. Biol. 139, 39–45 (2002).

    Article  CAS  Google Scholar 

  15. Dey, A., de With, G. & Sommerdijk, N. A. J. M. In situ techniques in biomimetic mineralization studies of calcium carbonate. Chem. Soc. Rev. 39, 397–409 (2010).

    Article  CAS  Google Scholar 

  16. De Jonge, N. & Ross, F. M. Electron microscopy of specimens in liquid. Nature Nanotech. 6, 695–704 (2011).

    Article  CAS  Google Scholar 

  17. Liao, H. G., Cui, L. K., Whitelam, S. & Zheng, H. M. Real-time imaging of Pt3Fe nanorod growth in solution. Science 336, 1011–1014 (2012).

    Article  CAS  Google Scholar 

  18. Yuk, J. M. et al. High-resolution EM of colloidal nanocrystal growth using graphene liquid cells. Science 336, 61–64 (2012).

    Article  CAS  Google Scholar 

  19. Parent, L. R. et al. Direct in situ observation of nanoparticle synthesis in a liquid crystal surfactant template. ACS Nano 6, 3589–3596 (2012).

    Article  CAS  Google Scholar 

  20. Radisic, A., Vereecken, P. M., Hannon, J. B., Searson, P. C. & Ross, F. M. Quantifying electrochemical nucleation and growth of nanoscale clusters using real-time kinetic data. Nano Lett. 6, 238–242 (2006).

    Article  CAS  Google Scholar 

  21. Li, D. et al. Direction-specific interactions control crystal growth by oriented attachment. Science 336, 1014–1018 (2012).

    Article  CAS  Google Scholar 

  22. Giuffre, A. J., Hamm, L. M., Han, N., De Yoreo, J. J. & Dove, P. M. Polysaccharide chemistry regulates kinetics of calcite nucleation through competition of interfacial energies. Proc. Natl Acad. Sci. USA 110, 9261–9266 (2013).

    Article  CAS  Google Scholar 

  23. Ihli, J., Bots, P., Kulak, A., Benning, L. G. & Meldrum, F. C. Elucidating mechanisms of diffusion-based calcium carbonate synthesis leads to controlled mesocrystal formation. Adv. Funct. Mater. 23, 1965–1973 (2012).

    Article  Google Scholar 

  24. Trotsenko, O., Roiter, Y. & Minko, S. Conformational transitions of flexible hydrophobic polyelectrolytes in solutions of monovalent and multivalent salts and their mixtures. Langmuir 28, 6037–6044 (2012).

    Article  CAS  Google Scholar 

  25. Wang, T., Zhao, C., Xu, J. & Sun, D. Enhanced Ca2+ binding with sulfonic acid type polymers at increased temperatures. Colloids Surf. A 417, 256–263 (2013).

    Article  CAS  Google Scholar 

  26. Verch, A., Gebauer, D., Antonietti, M. & Colfen, H. How to control the scaling of CaCO3: A “fingerprinting technique” to classify additives. Phys. Chem. Chem. Phys. 13, 16811–16820 (2011).

    Article  CAS  Google Scholar 

  27. Friedrich, H., Frederik, P. M., de With, G. & Sommerdijk, N. A. J. M. Imaging of self-assembled structures: Interpretation of TEM and Cryo-TEM images. Angew. Chem. Int. Ed. 49, 7850–7858 (2010).

    Article  CAS  Google Scholar 

  28. De Yoreo, J. J. & Vekilov, P. G. Reviews in Mineralogy and Geochemistry Vol. 54 57–93 (Mineralogical Society of America, 2003).

    Google Scholar 

  29. Hamm, L. M. et al. Reconciling disparate views of template-directed nucleation through measurement of calcite nucleation kinetics and binding energies. Proc. Natl Acad. Sci. USA 111, 1304–1309 (2014).

    Article  CAS  Google Scholar 

  30. De Jonge, N. & Ross, F. M. Electron microscopy of specimens in liquid. Nature Nanotech. 6, 695–704 (2011).

    Article  CAS  Google Scholar 

  31. Browning, N. D. et al. Recent developments in dynamic transmission electron microscopy. Curr. Opin. Solid State Mater. Sci. 16, 23–30 (2012).

    Article  CAS  Google Scholar 

  32. Evans, J. E., Jungjohann, K. L., Browning, N. D. & Arslan, I. Controlled growth of nanoparticles from solution with in situ liquid transmission electron microscopy. Nano Lett. 11, 2809–2813 (2011).

    Article  CAS  Google Scholar 

  33. Woehl, T. J. et al. Experimental procedures to mitigate electron beam induced artifacts during in situ fluid imaging of nanomaterials. Ultramicroscopy 127, 53–63 (2013).

    Article  CAS  Google Scholar 

  34. Noh, K. W., Liu, Y., Sun, L. & Dillon, S. J. Challenges associated with in-situ TEM in environmental systems: The case of silver in aqueous solutions. Ultramicroscopy 116, 34–38 (2012).

    Article  CAS  Google Scholar 

  35. Van de Put, M. W. P. et al. Writing silica structures in liquid with scanning transmission electron microscopy. Small (2014) 10.1002/smll.201400913

  36. Karuppasamy, M., Karimi Nejadasl, F., Vulovic, M., Koster, A. J. & Ravelli, R. B. G. Radiation damage in single-particle cryo-electron microscopy: Effects of dose and dose rate. J. Synchrotron Radiat. 18, 398–412 (2011).

    Article  CAS  Google Scholar 

  37. Jungjohann, K. L., Evans, J. E., Aguiar, J. A., Arslan, I. & Browning, N. D. Atomic-scale imaging and spectroscopy for in situ liquid scanning transmission electron microscopy. Microsc. Microanal. 18, 621–627 (2012).

    Article  CAS  Google Scholar 

  38. Stephens, C. J., Ladden, S. F., Meldrum, F. C. & Christenson, H. K. Amorphous calcium carbonate is stabilized in confinement. Adv. Funct. Mater. 20, 2108–2115 (2010).

    Article  CAS  Google Scholar 

  39. Tester, C. C. et al. In vitro synthesis and stabilization of amorphous calcium carbonate (ACC) nanoparticles within liposomes. CrystEngComm 13, 3975–3978 (2011).

    Article  CAS  Google Scholar 

  40. Nielsen, M. H., Aloni, S. & DeYoreo, J. J. In situ TEM imaging of CaCO3 nucleation reveals coexistence of direct and indirect pathways. Science 345, 1158–1162 (2014).

    Article  CAS  Google Scholar 

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We thank V. Altoe and S. Aloni for the use of, and assistance with, the JEOL-2100F, J. Tao for help with confocal Raman microscopy, and H. Friedrich and M. Nielsen for help with TEM data analysis. This research was supported by the US Department of Energy, Office of Basic Energy Sciences, at Lawrence Berkeley National Laboratory and at the Pacific Northwest National Laboratory (PNNL). Characterization of PSS globule formation was supported by the Materials Science and Engineering Division. Investigation of calcium carbonate nucleation was supported by the Division of Chemical Sciences, Geosciences, and Biosciences. Transmission electron microscopy was performed at the Molecular Foundry, Lawrence Berkeley National Laboratory, which is supported by the Office of Basic Energy Sciences, Scientific User Facilities Division. PNNL is operated by Battelle for the US Department of Energy under Contract DE-AC05-76RL01830. The work of P.J.M.S. and N.A.J.M.S. is supported by a VICI grant of the Dutch Science Foundation, NWO, The Netherlands.

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Authors and Affiliations



P.J.M.S. carried out most experiments and co-wrote the manuscript. K.R.C. provided expertise and support in the AFM measurements. P.J.M.S. and J.J.D.Y. performed the growth rate and diffusion analysis. R.G.E.K. contributed to developing and using the MATLAB procedure for growth rate determinations. N.A.J.M.S. and J.J.D.Y. designed the research and co-wrote the manuscript. All authors discussed the results and revised the manuscript.

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Correspondence to Nico A. J. M. Sommerdijk or James J. De Yoreo.

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Smeets, P., Cho, K., Kempen, R. et al. Calcium carbonate nucleation driven by ion binding in a biomimetic matrix revealed by in situ electron microscopy. Nature Mater 14, 394–399 (2015).

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