Our protocol describes a simple in vitro system, to study the effect of isolated molecules on the morphology and structure of calcium carbonate. This technique is advantageous in cases where the biomolecules I test are expensive, or available in small quantities, as well as when slow calcium carbonate precipitation is required. Furthermore, it allows to probe multiple precipitation experiments under the same conditions at once.
First, prepare the control experiment. Use triple distilled water and ethanol to clean glass pieces and glassware. Use a diamond pen to cut pieces of a glass microscope slide, so that they fit in a well of a 96-well plate.
Place the glass places in a beaker with triple distilled water, so that the water covers the glass slides. Sonicate in a bath sonicator for 10 minutes. Decant the water.
Add ethanol to cover the glass slides, and sonicate again in a bath sonicator for 10 minutes. Then, dry the slides and the glassware with a stream of nitrogen gas, and place them in an air plasma cleaner for 10 minutes at 130 watts. In a fume hood, fill the wells at the corners of a 96-well plate with ammonium carbonate powder, and seal the plate using aluminum foil.
Cover the foil with paraffin film. Clean any residual ammonium carbonate using nitrogen gas. Place the previously cut and cleaned glass pieces into five different wells closest to the center.
Fill each well bearing a glass piece with 100 microliters of calcium chloride solution prepared with triple distilled water at an increasing gradient of 10, 20, 30, 40, and 50 millimolar concentrations. Next, puncture the cover of each of the wells containing ammonium carbonate three times with a needle. Put the lid back, seal the borders with paraffin film, and keep it at 18 degrees Celsius in an incubator for 20 hours.
During the incubation, ammonium carbonate is decomposed into ammonia and carbon dioxide, which diffuse into calcium chloride solutions, resulting in the formation calcium carbonate crystals. After the incubation, open the lid carefully inside a fume hood, and use a loop to remove the crystals formed at the water-air interface. Use tweezers to transfer the glass pieces into a beaker containing double distilled water for a short dip.
Then, remove the samples from the beaker, and use double-sided tape to fix the glass pieces onto the bottom of the Petri dish. Dry excessive water touching the borders of the slide, with tissue wipes. Cover the Petri dish, and place it in a desiccator for 24 hours.
Observe the crystals formed on the glass pieces with an upright optical microscope at 10 to 40 times magnification. The observed rhombohedral crystals are most likely calcite. If in addition to the rhombohedral crystals, the solution contains spherical crystals which are most likely vaterite, repeat the crystallization protocol, making sure that the cleaning step is performed correctly.
Furthermore, make sure there is no ammonium carbonate in areas of the plate other than the dedicated wells. The optimal concentration for calcium chloride is determined according to the sample rich with smooth faceted calcite crystals without vaterite crystals. To begin, clean glass slides and glassware as done previously.
In a fume hood, place ammonium carbonate powder in the corners of a 96-well plate. In each well where precipitation will occur, place a glass piece that was cut and cleaned. Prepare the control wells.
Into two control wells, pipette 90 microliters of 25 millimolar Tris buffer at pH 8, supplemented with 100 millimolar sodium chloride. Then add 10 microliters of 0.5 molar calcium chloride stock solution. Then, adjust the concentration of additive protein TapA to 10 micromolar TapA, 100 millimolar sodium chloride, and 25 millimolar Tris buffer at pH 8.
Prepare the additive containing wells by adding 90 microliters of the additive solution. Add 10 microliters of 0.5 molar calcium chloride stock solution to the additive containing wells to reach the optimal concentration at 50 millimolar calcium chloride determined previously. Prepare the plate with holes on the cover over wells containing ammonium carbonate, and incubate at 18 degrees Celsius as previously done.
After incubation, prepare the glass pieces in a Petri dish as done before, and place the dish in a desiccator for 24 hours. To quantify the mass percentage of the additives in the calcium carbonate precipitates, first verify the extinction coefficient of the additive used. Then, use a microbalance to weigh the glass pieces where the crystals formed.
After that, scrape the crystals off the glass into an eppe tube with 1.2 milliliters of 0.1 molar acetic acid solution. Vortex briefly, and then place the tube into a sonicator to sonicate the sample until the crystals disappear. Store the sample at room temperature for 24 hours.
Weigh the glass slide after scraping off the crystals. Measure the UV absorbance A of the 1.2 milliliter sonicated solution at 280 nanometers for the protein additive. Use the Beer-Lambert equation to calculate its concentration c.
L is the optical path inside the cuvette. To calculate the mass of the additives in the crystal, use the equation, c times v equals m, if the concentration is in milligrams per milliliter. If the concentration is in moles per liter, then calculate the moles applying c times v equals n.
Then use the molecular weight to calculate the mass of the additives. Calculate the weight percentage of the additives in the crystals. M is the mass of the additives, and delta m s, is the mass of the calcium carbonate crystals that were scraped off the glass piece.
SEM images show the comparison between a proper control with smooth calcite faces, and calcite crystals with faces composed of steps. The spherical crystals are vaterite. The successful and unsuccessful control experiments resulted in Raman spectroscopy, showing the typical spectra of calcite and vaterite respectively.
The split of the Raman shift at around 1080 per centimeter is the most evident characteristic of vaterite. The crystals of calcium carbonate formed in the presence of TapA, are distinct from the control crystals. A complex spherical calcium carbonate assembly, composed of multiple calcite microcrystals was formed.
The Raman spectrum of the crystals formed in the presence of TapA, is similar to the spectrum of calcite. The absorbent spectrums of TapA, and the control without the additive, were measured following dissolution of the crystals in acid. The mass percent of TapA was determined to be 1.8 plus or minus 0.2%It's critical to follow the cleaning steps carefully, and to make sure that the control standards are met before testing the additives'effect on the formation of calcium carbonate.
In addition, it is important to remove any excess of ammonium carbonate in powdered form from the wells. Ammonium carbonate decomposes into ammonia and carbon dioxide. Ammonia is toxic if inhaled, and therefore, ammonium carbonate should be handled inside a fume hood only.
In order to evaluate the internal morphology and structure of the crystals, you can section them using a focused ion beam, image the sections with a transmission electron microscope, and measure the electron diffraction's patterns from specific locations along the sections. This technique has been previously used to study the effect of various molecules on the morphology and structure of calcium carbonate. We have elaborated this method to biopolymers that are produced by bacterial cells in biofilms.
It will be interesting to perform additional studies with other biopolymers produced by different bacterial strains.
