Local Light‐Controlled Generation of Calcium Carbonate and Barium Carbonate Biomorphs via Photochemical Stimulation

Abstract Photochemical activation is proposed as a general method for controlling the crystallization of sparingly soluble carbonates in space and time. The photogeneration of carbonate in an alkaline environment is achieved upon photo‐decarboxylation of an organic precursor by using a conventional 365 nm UV LED. Local irradiation was conducted focusing the LED light on a 300 μm radius spot on a closed glass crystallization cell. The precursor solution was optimized to avoid the precipitation of the photoreaction organic byproducts and prevent photo‐induced pH changes to achieve the formation of calcium carbonate only in the corresponding irradiated area. The crystallization was monitored in real‐time by time‐lapse imaging. The method is also shown to work in gels. Similarly, it was also shown to photo‐activate locally the formation of barium carbonate biomorphs. In the last case, the morphology of these biomimetic structures was tuned by changing the irradiation intensity.

. UV-Vis absorbance spectrum of Ketoprofen 20 mM in water at pH 8.9

Optimization of the photoreactive solution for the photo-precipitation of calcium carbonate
For local irradiation, we used the light from a 365 nm UV LED in a spot with a 300 µm radius (irradiance 1.3 mW/mm 2 ) in a closed cell with 0.1 mm thickness and 15x25 mm 2 size, as schematized in the main text, figure 1. The cell was filled with a Ksolution of 20 mM at pH = 8.9. and was irradiated for 120 s; during irradiation, the transmission images were acquired with a 20 MPX color CMOS camera by trans-illuminating a large area (about 4x4 mm 2 ) of the cell, centered on the UV spot, with white light (not absorbed by K -).

Demonstrating local photo-precipitation of PP
The local irradiation of the sample mentioned above led to the formation of oily droplets of the PP. The chemical nature of the PP was investigated by gas chromatography-mass spectrometry (see below), in which a single chromatographic peak characteristic of 3-ethylbenzophenone was observed. The precipitation of the PP creates a double problem. First, because of their size, these droplets diffused very slowly outside the irradiation area, remaining in the illumination spot even 30 min of illumination (see Figure S2 a), filtering the UV light, limiting the photoreaction rate, and reducing the generation of bicarbonate.

Demonstrating photo-generation of HCO3 -
To demonstrate the photogeneration of HCO3 -, we irradiated with the 365 nm LED a 20 mM solution of Kat pH = 8.9 (obtained by the addition of NaOH) in a closed vial measuring in the meantime the pH with a glass electrode. As shown in figure S2 (black dots), a decrease in the pH was observed during irradiation. Nevertheless, this change was relatively small because of the screening effect mentioned above.

Preventing local photo-precipitation of PP
To avoid the PP precipitation, the experiments were repeated in the presence of Triton X-100  (black dots) a solution containing Kat initial pH 8.9 (adjusted with NaOH); (red triangles) a solution containing Kat initial pH 8.9 (adjusted with NaOH) and Triton X-100 0.3 mM; (green squares) a solution containing K -(NH4OH/NH3 buffer, pH=8.9) and Triton X-100 0.3 mM c) Transmission image obtained in the same conditions as for b) in the presence of Triton X-100 0.3 mM.

Preventing pH drop during the photoreaction
While the solution acidification is a clear indicator of the formation of HCO3 -, it does not favor the aim of using this photoreaction to precipitate calcium carbonate. The solubility of calcium carbonate is strongly pH-dependent, and it increases at lower pH values. Therefore, an alkaline pH must be secured to trigger the precipitation. Hence, an NH4OH/NH3 buffer (40 mM, pH 8.9) was added to prepare a solution containing K -20 mM and Triton X-100 (0.3 mM). In this latter solution, as shown in figure S2 b (green squares), a minor pH change was observed during the irradiation.

Mass Spectrum of PP
Electronic impact mass spectrometry (GC-MS) was performed on Ketoprofen 6 mM solution in water/ethanol 1:1 mixture after 1 hour of LED irradiation ( Figure S3). The LED was placed horizontally, at 2 cm from the vial.

Details for pH measurements
The pH change of Ketoprofen (20 mM) solution in water and water with Triton X-100 0.3 mM was monitored during LED irradiation. The LED was placed horizontally, at 2 cm from the vial.
To estimate CO2 production effect on the overall pH, monitoring was performed irradiating Ketoprofen (20 Mm) solution at pH 9 in two cases: in the presence of NaOH (in water and in water and Triton X-100 0.3 mM) and in the presence of NH3 (in water and Triton X-100). The change in pH was monitored every 3 minutes for 15 minutes.

Dynamic Light Scattering (DLS) of Triton X-100 and Ketoprofen solution
The size of the nanoparticles formed in the presence of the surfactant, Triton X-100, was investigated via DLS. In particular, a solution of 20 mM Ketoprofen and 0.3mM Triton X-100 in water at pH=9 was prepared, and the analysis was performed in two different time intervals using PMMA semi-micro cuvettes. The DLS was received after irradiating the solution for 15 ( Figure S5b) and 25 minutes with the 365nm LED. The results indicated the presence of nanoparticles with a size around 100nm and narrow size distribution ( Figure S5a). and dried. SEM measurements were performed with a Hitachi TM3000 and are shown in Figure S5.
In Figure S5, it can be noted that a lot of Calcite crystals are deposited on the organic matrix, which probably corresponds to the water-insoluble Ketoprofen by-product formed after irradiation. Figure S5. SEM images of CaCO3 deposited on organic matrix after laser irradiation at 355 nm

Photoreaction quantum yields
Quantum yields ( Figure S5) were calculated by irradiation of Ketoprofen 20 mM in water, without and with CaCl2 20 mM. [8] Irradiation was performed in a fluorimeter (Fluoromax) at 365 nm, with a slit of 20 nm. Almost 3 mL of solution were irradiated under stirring; every 10 minutes, the solution was centrifuged (5 min, 8000 rpm) and filtered twice with a 0.45 µm PVDF filter. Then the absorption spectrum was measured before continuing with the irradiation. The photoreaction quantum yield was calculated based on the Ketoprofen absorbance decrease at 345 nm. This value, which is in the tail of the absorption band of Ketoprofen, whose maximum is at 270 nm, [7] was chosen because the high Ketoprofen concentration did not allow to monitor its absorbance variation at the maximum of the peak. As Figure S5 shows, the quantum yield does not depend on the presence of Calcium Chloride and is 1,5%.

Gel preparation
Agarose (20 mg/mL) is added to a sucrose 60% w/v aqueous solution, in presence of CaCl2 20 mM.
The system is heated at 85°C to dissolve agarose. The hot solution is left to cool down between two glass slides to obtain a very smooth and uniform gel with a thickness of 1 mm.

Photo-precipitation of Calcium carbonate embedded in a gel
A piece of the gel prepared with CaCl2 20 mM (almost 1cmx1cm) was placed in a disposable well (0.57 cm 2 ) and covered by 350 µL of Ketoprofen 20 mM solution at pH 9.6. The irradiation is performed using a LED at 365 nm focused by the fluorescence microscope, utilizing a 4x objective.
Then, the solution is removed, and the gel is washed with MilliQ water, which is then removed. The formation of crystals in the gel is then observed by the camera, with the 4x objective.

Silica-Carbonate Biomorphs
Initially, a solution of NaOH 2.3M was prepared by dissolving 90mg of solid NaOH in 1ml of water while degassing with N2. A solution of Ketoprofen (10Mm) was prepared in a vial by diluting 25mg of Ketoprofen in 10ml of water and was solubilized by adding 150μl of the previously-prepared NaOH solution together with 30μl Sodium Silicate. The mixture was degassed for 10 minutes.
Simultaneously, a 10mM BaCl2 solution was prepared and was also degassed. Following, 0.5ml of the Ketoprofen-Silicate solution was mixed with the same amount of BaCl2 solution (0.5ml) and 2μl of Triton X-100, and the final solution was degassed for 5 minutes.
Then, 20μl of the degassed Ketoprofen-Sodium Silicate-BaCl2 solution were placed in the irradiation cell made by two slides of glass separated by a two layers of adhesive tape as spacers and covered with Baysilone-Paste (GE Bayer Silicones) to have a shielded environment. The current intensity was adjusted at 0.1 and 0.9 A to observe the process induced by two different light intensities. The acquisition was performed for one hour, focusing with the 10X objective and receiving a frame every 10 seconds. After one hour, images with 60X magnification (objective Olympus UPLFLN, 60X) were also collected for the observation of the morphology of the biomorphs.