Calcium Carbonate Formation in the Presence of Biopolymeric Additives

Summary

We describe a protocol for the precipitation and characterization of calcium carbonate crystals that form in the presence of biopolymers.

Abstract

Biomineralization is the formation of minerals in the presence of organic molecules, often related with functional and/or structural roles in living organisms. It is a complex process and therefore a simple, in vitro, system is required to understand the effect of isolated molecules on the biomineralization process. In many cases, biomineralization is directed by biopolymers in the extracellular matrix. In order to evaluate the effect of isolated biopolymers on the morphology and structure of calcite in vitro, we have used the vapor diffusion method for the precipitation of calcium carbonate, scanning electron microscopy and micro Raman for the characterization, and ultraviolet-visible (UV/Vis) absorbance for measuring the quantity of a biopolymer in the crystals. In this method, we expose the isolated biopolymers, dissolved in a calcium chloride solution, to gaseous ammonia and carbon dioxide that originate from the decomposition of solid ammonium carbonate. Under the conditions where the solubility product of calcium carbonate is reached, calcium carbonate precipitates and crystals are formed. Calcium carbonate has different polymorphs that differ in their thermodynamic stability: amorphous calcium carbonate, vaterite, aragonite, and calcite. In the absence of biopolymers, under clean conditions, calcium carbonate is mostly present in the calcite form, which is the most thermodynamically stable polymorph of calcium carbonate. This method examines the effect of the biopolymeric additives on the morphology and structure of calcium carbonate crystals. Here, we demonstrate the protocol through the study of an extracellular bacterial protein, TapA, on the formation of calcium carbonate crystals. Specifically, we focus on the experimental set up, and characterization methods, such as optical and electron microscopy as well as Raman spectroscopy.

Introduction

Biomineralization is the formation of minerals in the presence of organic molecules, often related with functional and/or structural roles in living organisms. Biomineralization may be intracellular, as in the formation of magnetite inside magnetotactic bacteria¹, or extracellular, as in the formation of calcium carbonate in sea urchin spikes², of hydroxyapatite that is related with collagen in bones³ and of enamel that is associated with amelogenin in teeth⁴. Biomineralization is a complex process that depends on many parameters in the living organism. Therefore, in order ....

Protocol

1. Calcium carbonate crystallization

1. Control preparation and optimization
   1. Prepare clean glass pieces. Use the same cleaning procedure to clean the glassware.
      1. Use a diamond pen to cut pieces of a glass microscope slide so that they fit in a well of a 96-well plate.
         NOTE: 5 mm x 5 mm pieces should largely fit.
      2. Place the glass pieces in a beaker with triple distilled water (TDW) so that water covers the glass slides and sonicate in a bath sonicator for 10 min.
      3. Decant the water, add ethanol to cover the glass slides, and sonicate in a bath sonicator for 10 min.
      4. Dry the slid....

Results

A schematic of the experimental set up is shown in Figure 1. Briefly, the diffusion method is used in order to form calcium carbonate crystals in 96-well plates and test the effect of biopolymers on the morphology and structure of the calcium carbonate crystals. In these experiments, ammonium carbonate is decomposed into ammonia and CO₂, which diffuse into calcium carbonate solutions, resulting in the formation of calcium carbonate crystals (Figure 1 .......

Discussion

The method described here is aimed at forming calcium carbonate crystals in the presence of organic additives and evaluating the effect of organic biopolymers on the morphology and structure of calcium carbonate crystals in vitro. The method is based on the comparison of the crystals formed in the presence of the organic additives to the calcite crystals formed in the control experiment. We have shown how to use the diffusion method to form the calcium carbonate crystals, how to characterize their morphology using optica.......

Materials

Name	Company	Catalog Number	Comments
Acetic acid	Gadot	64-19-7
Ammonium carbonate	Sigma-Aldrich	506-87-6
Calcium chloride dihydrate	Merck KGaA	10035-04-8
Ethanol Absolute	Gadot	64-17-5
Micro-Raman	Renishaw		inVia Reflex spectrometer coupled with an upright Leica optical microscope
Microscope	Nikon		Eclipse 90i model
Nis elements Br software	Nikon		For microscope imaging
Scanning Electron Microscope	ThermoFisher Scientific		FEI Sirion microscope
Spectrophotometer	JASCO		V-670 model
Sputter coater	Polaron		SC7640 model