Biomimetic Mineralization of Calcium Carbonate in Aqueous Biphasic Systems

Open Access
Author:
Cacace, David Neal
Graduate Program:
Chemistry
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
December 05, 2013
Committee Members:
  • Christine Dolan Keating, Dissertation Advisor
  • Philip C. Bevilacqua, Committee Member
  • Scott A Showalter, Committee Member
  • James Hansell Adair, Committee Member
Keywords:
  • Aqueous two-phase systems
  • calcium carbonate
  • intermediate chelation
  • mineralization vesicles
Abstract:
Many organisms form minerals with unique morphologies and specialized functions through the intercellular process of biomineralization. In particular, calcium carbonate (CaCO3) biomineralization often begins within mineralization vesicles. It is theorized that the binding and control over free Ca2+ concentrations both in the cytoplasm and mineralizing vesicles are in part responsible for the resulting CaCO3 products. The goal of this dissertation is the generation of a biomimetic mineralization vesicle system that controls Ca2+ binding in tandem with local CaCO3 precipitation towards understanding similar interdependencies in biological analogs. In Chapter 1, I provide a detailed introduction to serve as a framework for the rest of the Chapters. Chapter 2 is focused on large scale (~20 mL) aqueous two-phase systems (ATPS) of poly(ethylene glycol) (PEG) / dextran (Dx), where small cations such as Ca2+ are added to observe their partitioning behavior. The polyanion dextran sulfate (DxS) is also added to this system to strongly affect Ca2+ partitioning and allow for localized CaCO3 precipitation, although this method was not used in Chapters 3-5. Chapter 3 also used the large scale PEG/Dx ATPS for the localized precipitation of CaCO3, which was the result of a compartmentalized enzyme. This enzyme, urease, was localized mainly in the Dx-rich phase, and released CO3 2- when its substrate (urea) was introduced. The effect of PEG/Dx volume ratios on the CaCO3 precipitation rate was investigated, as well as any morphological or polymorphic changes that occured due to the presence of the high concentration of macromolecules. In biological cells, compartmentalization is thought to iv have a direct impact on the local rate of various biological processes, especially concerning compartmentalization of Ca2+ within mineralization vesicles. Chapters 4 and 5 detailed the formation of stabilized ATPS emulsion droplets using large unilamellar vesicles (LUVs), which resulted in Dx-rich droplets of ~5 μm diameter. Here the enzyme urease continued to compartmentalize into the Dx-rich droplets, however added Ca2+ caused the lipids to aggregate. This was corrected for using a number of Ca2+ chelators with varying binding strength. Chapter 4 and Chapter 5 investigated the use small and polymeric Ca2+ chelators, respectively. In both Chapters, the LUVs were stabilized and CaCO3 precipitated when Ca2+ was chelated properly. In Chapter 5, the chelator used introduced a new polymer-rich phase that precipitated entirely amorphous calcium carbonate (ACC) when the enzymatic reaction proceeded. This is of biological relevance because biomineralizing organisms also selectively precipitate ACC within mineralizing vesicles prior to other polymorphic or morphological changes. This dissertation presents results that mimic some of the conditions that occur in actual biomineralizing organisms. In the future, this system could be used with chelators extracted from organisms or be redesigned to precipitate different types of biominerals to elucidate some of the underlying chemical principles that govern biomineralization.