Electrocrystallization of CaCO₃ Crystals Obtained through Phosphorylated Chitin

Abstract

A phosphorylated chitin (Chi-P) derivative was synthesized and its chemical structure was verified with Fourier-transform infrared spectroscopy (FTIR), elemental analysis, and thermogravimetric techniques (TGA). The influence of Chi-P used as a solid template through in vitro electrocrystallization (EC) supported on an indium zinc oxide (ITO) surface on the growth of calcium carbonate (CaCO₃) was studied. CaCO₃ crystals through EC essays were also compared with crystals obtained with the gas diffusion (GD) method. Scanning electron microscopy (SEM), energy dispersive X-ray spectrometry (EDS), chronopotentiometry, Raman, and powder X-ray diffractometry (XRD) characterized all resultant inorganic particles. Our findings revealed that the EC method selectively controlled the coexistence of truncate calcite and the metastable phase of vaterite. The crystals’ morphology reflects the electrostatic interaction of phosphate moieties from Chi-P onto CaCO₃ crystals through both EC and GD crystallization methods. We believe that the EC method represents a viable electrochemical approach for studying different inorganic minerals and could be useful as an in vitro classical crystallization method for the design of advanced inorganic materials with desirable shapes and properties.

1. Introduction

Biological crystallization is the process by which living organisms produce hybrid biogenic composites and exert accurate control over the minerals they deposit, creating advanced materials with uniform particle size, novel morphology, and myriad shapes or sizes that often exhibit high strength and remarkable properties [1,2,3,4]. It is known that the inorganic crystallization mechanism is altered by specific interactions between chemical groups, such as –CO₂H, –PO₃H, –SO₃H containing insoluble additives or functionalized networks as solid templates [5,6,7,8,9,10]. In this sense, calcium carbonate (CaCO₃) represents the most abundant biomineral in nature and it has considerable industrial interest. It nucleates in three known crystalline polymorphs: calcite, aragonite and vaterite [11,12,13]. For understanding the biological control of CaCO₃ crystallization many studies regarding the effect of natural and synthetic polymers, copolymers, functionalized biopolymers, and adsorption surface phenomena involved in this process have been reported [14,15,16,17,18,19,20]. In order to achieve control over this biomineral it is important to understand the mechanisms of how crystals interacts between organic and inorganic components using different classical crystallization setup in a similar manner that they occur in nature. Such selective control is governed by different functionalized biomolecules, including proteins, polysaccharides, glycosaminoglycan, etc. Therefore, control of polymorphs of CaCO₃ is strongly affected by additives chemical groups (e.g., carboxylic, phosphate), crystallization method, and precipitation conditions (e.g., pH, concentration, temperature, etc.) [21,22,23,24,25]. Sulfate groups in biopolymers and phosphate groups of proteins have been known as active groups on biomineralization of hard bioceramic exoskeletons and eggshell calcified layer [8,26]. We expected that functionalized biopolymers like phosphorylated chitin (Chi-P) used as a solid template could be an efficient crystallization modifier acting by adsorption onto the active growth sites of the CaCO₃ surface under the biopolymer-controlled crystallization concept. It is well known that chitinous and collagenous components represent the main structural skeletal system, which support the cellular structure of abundant animal tissues with active roles on biological mineralization [27,28]. Moreover, non-functionalized chitin (Chi) in the structural and shell formation at in vitro level has been described as a non-mineralized laminar substrate or framework, which compartmentalizes a microenvironment where mineralization take place [29].

On the other hand, gas diffusion (GD) crystallization has been widely employed to study the effect of a number of ionic macromolecules (proteins, polysaccharides, surfactants) and biological organic support (egg-shell matrix, gastrolith) on CaCO₃ crystallization [30,31,32,33,34,35,36,37]. Meanwhile, the electrocrystallization (EC) method, as an electrochemical concept approach, represents a novel crystallization technique of inorganic materials, such as calcium salts (carbonate, sulfate, oxalate, etc.). It is important to note that the EC method has some technical advantages, e.g., control of applied voltage and current, concentration of additives, times that are precisely controlled inducing selective control of the type of crystals grown onto the indium zinc oxide (ITO) substrate [38,39,40,41], etc. In this regard, our group has recently extracted and purified alginate from Chilean algae (Lessonia nigrescens), and tested the plant extract from Dendroligotrichum dendroide polytrichaceae (Chilean family) as an additive on CaCO₃ and calcium oxalate (CaOx) through EC on electrospun polycaprolactone fibers [42].

The aim of this work was to study the influence of phosphorylated chitin (Chi-P) on CaCO₃ particles through EC method supported onto a conductive ITO glass surface, and the obtained crystals were compared to those obtained with the gas diffusion (GD) method. This study was supported and performed in the context of the active anionic groups on biomineralization, low solubility of Chi-P in acid form, and the scarce knowing effect of Chi derivatives on in vitro CaCO₃ crystallization.

2. Materials and Methods

2.1. Reactants

Calcium chloride dihydrate (CaCl₂·2H₂O), sodium bicarbonate (NaHCO₃), sodium hydroxide (NaOH), sodium chloride (NaCl) and potassium nitrate (KNO₃), methanesulfonic acid and phosphorous pentoxide were obtained from Merck (Kenilworth, NJ, USA). All solvents and other reagents were of the highest available purity. Indium zinc oxide (ITO) conductive glass electrodes were used as the conductive material for electrocrystallization of CaCO₃ and were purchased from Delta Technologies (Dallas, TX, USA). ITO working electrodes were purchased from Corning^® and aluminosilicate glass, 25 × 25 × 1.1 mm, coated with one surface of RS = 5–15 Ω was used. The chitin sample was obtained in the laboratory by standard demineralization and deproteinization processes of Chilean red prawn (Pleuroncodes monodon) exoskeletons [43]. The synthesis of P-Chi is described as is shown in Appendix A (please see Appendix A.1).

2.2. Gas Diffusion (GD) Method

GD crystallization of CaCO₃ particles was carried out in the presence of Chi-P as polymeric solid template and compared in the absence of Chi-P as negative control at room temperature for 24 h. GD method was performed as we described in our previous works [37,44,45,46,47,48].

2.3. Electrocrystallization (EC) Method

In vitro EC of CaCO₃ particles were produced in the presence of Chi-P onto the ITO glass surface at room temperature using a galvanostat/potentiostat BASi Epsilon (West Lafayette, IN, USA) instrument. The chronopotentiometry technique was used during the EC of CaCO₃. ITO were washed with methanol and Milli Q water, obtained from a water purification system LaboStar 4-DI/UV and sonicated for 5 min. CaCO₃ crystallization by using electrolyte solution with dissolved O₂ in alkaline media was carried out through an electrochemical reaction. The electrolytic solution consisted of 0.2 mM CaCl₂, 6 mM NaHCO₃, and 10 mM NaCl. The EC equations of EC of CaCO₃ has been as previously described [39]. The utilized reactants concentrations of the EC of CaCO₃ demonstrated clearly the production of CaCO₃ particles on ITO substrate. Before the EC of CaCO₃ essays, a linear sweep voltammetry was performed in order to determine first the maximal potential value where the reduction of molecular O₂ is reached. The maximum voltage activity established was at −890 mV [49]. Therefore, the use of this potential value generate a basic pH media allowing efficient conversion of bicarbonate to carbonate ions favoring the CaCO₃ particle formation. Before the EC of CaCO₃ essays was performed, the electrochemical cell was saturated with molecular O₂ for 40 min. After the above potential was fixed, a set of chronoamperometry tests on CaCO₃ coating for 120 min was carried out.

2.4. Characterization

The surface morphology of the resultant CaCO₃ crystals was characterized by scanning electron microscopy (SEM) using JEOL JSM-6390LV and HITACHI TM3000 instruments. Fourier transform infrared spectroscopy (FTIR), and attenuated total reflectance in conjunction with FTIR (FTIR-ATR) analysis were performed by using FTIR Nicolet Magna 5PC spectrophotometer coupled to a PC with OMNIC software to data analysis and a Interspectrum Interspec p/n 200-X spectrometer (Toravere, Estonia) was used. TGA of Chi and Chi-P was carried out using a Netzsch (Selb, Germany), model STA 409 PC, system. The sample in a platinum crucible was heated from 25 °C to 800 °C at 10 °C/min in air atmosphere (70 mL/min). The XRD diffraction spectra were taken from a powdered resultant CaCO₃ sample using a Siemens D-5000 X-ray diffractometer.

3. Results

In order to evaluate the Chi-P effect as a solid template on in vitro CaCO₃ mineralization, a set of CaCO₃ crystallization essays using the EC method were performed and the morphology of the resultant CaCO₃ particles deposited onto the Chi-P surface were compared with the crystals obtained through GD method. The illustration of the EC of CaCO₃ on the Chi-P and experimental conditions can be observed in Figure 1.

The EC of CaCO₃ method is an electrochemical electrodeposition technique of inorganic material based on the use of a specific voltage in an electrochemical solution media that induces the formation of crystals in a controlled manner. The EC method consists of two stages: first, the particles suspended in a solution are forced to move towards an electrode by the application of a certain electric field, and then inorganic particles accumulate and deposit on the conductive ITO glass surface as a working electrode (WE). Once the EC tests were carried out, it was determined that CaCO₃ crystals can be generated on ITO substrates in the absence and presence of Chi-P. This is due to the highly conductive capacity of the ITO substrate, which conducts the current previously established in the program at −890 mV in the electrochemical solution, allowing the reduction of oxygen, which generates an increase in the pH value that favors the conversion of bicarbonate to carbonate [39]. It is well known that the ITO substrate represents a conductive material with optoelectronic applications due to its good optical transparency, high conductivity, and electrochemical stability [40]. CaCO₃ crystals obtained with EC method in the presence and absence of Chi-P onto ITO substrate were compared and evaluated through the measurement of current (mA) at different times (min). For this purpose, an electrolytic solution was placed into an electrochemical cell and the EC essays were performed in a galvanostat/potentiostat BASi Epsilon instrument. Figure 2 shows the chroamperometric curves related to crystalline deposits of CaCO₃ onto the ITO surface during the EC performed on the ITO by using −890 mV for 120 min.

Figure 2 shows a notorious decrease in the electrochemical current curve during the EC of CaCO₃ when Chi-P was used as a template (Figure 2B), in a similar manner than the control experiment without Chi-P (Figure 2A). However, the initial induced current was higher in Chi-P (ca. 1.0 mA) than control (ca. 0.42 mA). Moreover, we found the generated current stabilization for Chi-P begin ca. of 2.10 min at 0.11 mA instead of ca. 0.12 min at 0.17 mA in the absence of the additive. In both cases, the applied current became asymptotic until the end of the EC experiment at 0.035 mA and 2.15 min, respectively.

In order to evaluate the morphogenetic effect of Chi-P template on CaCO₃ crystals growth onto ITO surface a scanning electron microscopy (SEM) was performed. Figure 3 shows the SEM images of CaCO₃ crystals obtained on Chi-P surface. In general, Figure 3A shows the typical rhombohedral calcite crystals growth on ITO in absence of additive (control) of size ca. 10 μm. Figure 3B–D shows abundant truncate and rounded calcite particles growth in the presence of Chi-P as solid template of size ca. 8–20 μm. In addition, swollen and extended Chi-P sample embedded into the electrolytic solution and the starting rigid Chi-P film sample before the EC essays was observed in the Figure 3E,F, respectively. Then, diffusion of Chi-P molecules at the local ITO surface may have tremendous influence on the local nucleation of CaCO₃ crystals, or alter the process of crystal formation as we observed by energy dispersive X-ray spectrometry (EDS) analysis.

It is well known that scanning electron microscopy/energy dispersive X-ray spectrometry (SEM/EDS) is a widely applied elemental microanalysis technique capable of identifying and quantifying almost all elements in the periodic table. With this in mind, SEM-EDS measurements of the resultant modified CaCO₃ particles’ growth onto the ITO surface in the presence of Chi-P, Chi-P and ITO surfaces were conducted and all results are shown in Figure 4.

The ATR/FTIR technique allows that samples, in liquid or solid matter state, be examined directly without further preparation. In order to determine the chemical composition of Chi-P film (starting sample) and the resultant CaCO₃ crystals precipitated through EC on Chi-P surface we used attenuated total reflectance (ATR) and Fourier-transform infrared spectroscopy (FTIR) as a sampling technique to provide excellent quality data in conjunction. The FTIR analysis is described in Appendix A (please see Appendix A.2). Figure 5 shows the FTIR spectra of Chi-P film, commercial CaCO₃ and deposited CaCO₃ particles. The spectra of Chi derivative plus CaCO₃ crystals is slightly different to the original Chi-P spectrum showing new absorption bands at 1390 cm⁻¹ and 871 cm⁻¹, respectively. They are coincident with the broad band that appears in commercial CaCO₃ samples at 1394 cm⁻¹ (υ₃ asymmetric CO₃²⁻) and with the sharp band at 871 cm⁻¹ (υ₂ asymmetric CO₃²⁻). Those bands confirmed the presence of deposited CaCO₃ crystals.

In addition, the ATR/FTIR analysis was based on the presence of new absorption bands associated to CaCO₃ deposition on the Chi-P surface. The inorganic particles depositions was confirmed through the absorption bands representing the main vibrations of the carbonate ions (CO₃²⁻). For a more detailed analysis of samples, the ATR/FTIR spectra for Chi-P and Chi-P + CaCO₃ in different spectral regions are obtained (Figure 6).

In Figure 6 the FT-IR spectra of Chi-P and Chi-P + CaCO₃ were shown. The main absorptions of CO₃²⁻ ions were analyzed and it is important to note that most of its characteristics vibration bands appear only in the Chi-P + CaCO₃ spectra. This suggests that spectral changes for this sample are associated with salt deposition on the Chi-P surface. In Figure 6A the weak vibrational band observed at 1073.5 cm⁻¹ in Chi-P + CaCO₃ spectra only was assigned to CO₃²⁻ (symmetric stretching, ν₁) from vaterite crystalline polymorph. Figure 6B shows two carbonate bands seen at 876.2 cm⁻¹ that are common to both calcite and vaterite (asymmetric deformation, ν₂) and at 850.4 cm⁻¹ (ν₂, vaterite), respectively. The absorption band at 711.1 cm⁻¹ is characteristic for calcite, whereas in vaterite the same band (symmetric deformation, ν₄) is shifted to 743.3 cm⁻¹ (Figure 6C) [50]. Previous research found that latter vibrations are characteristic of crystalline CaCO₃ phases only [51,52]. In Figure 6D the main CO₃²⁻ bands at 1429.8 cm⁻¹ and 1424.3 cm⁻¹ (asymmetric stretching, ν₃) for vaterite and calcite polymorphs are presented, respectively. From previous analysis it could be concluded that a mixture of two CaCO₃ crystalline polymorphs, vaterite and calcite, were crystallized on the surface of the Chi-P membrane. Those results are consistent with XRD analysis.

In order to further study the relationship between CaCO₃ and Chi-P, a detailed FTIR analysis of Chi-P and Chi-P + CaCO₃ spectra in the PO₄³⁻ absorption regions are presented in Figure 7.

From Figure 7 it could be observed that the presence of deposited CaCO₃ salt changes the shape of some bands in FTIR spectra, producing phosphate bands that are more resolved than in the original Chi-P spectra. This fact could be due to different chemical environments for those PO₄³⁻ groups from the Chi-P membrane surface involved in the interaction with Ca²⁺ ions at deposition sites to form calcium phosphate or its hydrogen phosphate salts. This is supported by the presence of O-P-O bending bands v₄ PO₄³⁻ (572.9 cm⁻¹, 558.5 cm⁻¹, 600.1 cm⁻¹) and HPO₄²⁻ (551.2 cm⁻¹) (Figure 7A). Other corresponding characteristic bands (P-O stretching of P-OH) are v₁ PO₄³⁻ (953.2 cm⁻¹), v₃ PO₄³⁻ (1035.28 cm⁻¹, 1077.8 cm⁻¹) (Figure 7B). All those bands are coincident with those previously reported for calcium phosphate salts, demonstrating its formation on the surface of the Chi-P membrane [53,54,55,56].

In order to determine the internal lattice of crystalline CaCO₃ products obtained through EC method and to determine the polymorphism of resultant CaCO₃ crystals growth on Chi-P onto ITO substrate, X-ray diffraction (XRD) technique was performed as we can observe in Figure 8. Figure 8 shows the XRD pattern of CaCO₃ crystals obtained by the EC method (Figure 8A), compared to the spectrum of the starting Chi-P sample (Figure 8B).

4. Discussion

4.1. Chemical Characterization of Chitin (Chi) and Chitin Phosphate (Chi-P)

The ATR spectra of starting Chi and functionalized Chi-P samples can be observed in the Supplementary Materials Figure S1. The typical amide I band at about 1655 cm⁻¹ and amide II band at about 1555 cm⁻¹ that gives the acetyl content in Chi are present. A broad 3000–3200 cm⁻¹ band due to N–H and O–H stretching with small shoulders at about 3106 cm⁻¹ band is observed for this biopolymer. From the spectrum of Chi-P several differences are apparent when compared with the spectrum of former lobster Chi. The peak at 1380 cm⁻¹ was attributed to P=O stretching and the peak at 1042.7 cm⁻¹ was due to –P–OH groups from Chi-P. The presence of a small peak at 956.1 cm⁻¹ may also be attributed to the P–O stretching of P–OH, overlap with CH₃ rocking band after Chi phosphorylation [57]. Furthermore, the Chi peak from O–H deformation of –CH₂–OH at 1378 cm⁻¹ almost disappears. It seems that Chi primary hydroxyl groups are involved in the substitution reaction as expected.

The thermal properties of lobster Chi and Chi-P were also investigated by TGA and results are given in Figures S2 and S3. It is important to clarify that the TGA was used only to demonstrate the phosphorylation reaction of chitin as supporting material. The resultant inorganic mineral on Chi-P was not considered in the current work by TGA due to the hybrid CaCO₃-Chi-P material was demonstrated by using spectroscopic techniques (FTIR, Raman), X-ray diffraction (XRD), chronoamperometric curves and SEM analyses. The thermogravimetric (TG) curves from both compounds shown a first mass loss in the range of 55–110 °C due to water evaporation. The TG curve of Chi shows two maximum decomposition rates peaking at 316.4 °C and 384.1 °C, which are associated to weight losses of 12.1 and 60.2%, respectively. The first effect was attributed to a complex process corresponding to the initial biopolymer pyrolysis stage. During this process, simultaneous ring dehydration, depolymerization, and decomposition of deacetylated units of the polysaccharide occurs. In the second and larger process decomposition of acetylated units occurs, including polymer deacetylation. The Chi decomposition temperature range observed was in agreement with previously reported for other Chi sources between 300–460 °C [58]. On the contrary, it could be observed that Chi-P shown a complex decomposition process having three weight losses, which is a typical behavior of block copolymers. The maximum decomposition rates for each stage was peaking at 243.5 °C, 316.4 °C, and 480.1 °C and were associated to weight losses of 20.8, 18.3, and 3.1%, respectively. The first decomposition stage could be related to thermal decomposition of phosphorylated units where the hydrolysis of phosphate groups linked to Chi backbone occurs provoking a polymer backbone hydrolysis and dehydration. The second stage is assigned to Chi units decomposition processes, taking in count the coincidence in maximum decomposition rate temperature among both samples. It is of interest to note that the third stage occurs at 96 °C higher temperature than in Chi. It could be due to thermal degradation of residual compounds resulting from previous thermal stages (e.g., crosslinked chains) or formation of new type of phosphate species but this subject will deserve further investigation. In general, Chi-P was less thermal stable than Chi, which is in agreement with previous report for other Chi phosphate derivatives [57]. Finally, the elemental analysis allows us to determine the Chi-P degree of substitution that was 0.38 from the P/N mol ratio.

4.2. Study of CaCO₃ Crystals Deposition by EC and GD Methods

The GD method of CaCO₃ as in vitro crystallization has been widely employed using different biomacromolecules_, instead of the EC method, which uses an electrochemical approach, can be realized in a short time and the resistant inorganic material selectively growth on the ITO substrate can be also directly monitored by using chronoamperometry or chronopotentiometry analysis. One of the aim of this work was to use both crystallization methods for knowing the effect of the same biomolecule using as additive or electrodeposited on the ITO conductive support as a solid template. It is well known that anionic functionalization, e.g., carboxylic, sulfate, or phosphate groups attach to the main chain of macromolecules has been reported as active group on biomineralization. Therefore, the EC method was selected to study the effect of Chi-P on CaCO₃ and the resultant crystals compared to the classical GD method. The Elemental analysis is described in Appendix A (please see Appendix A.3).

4.3. Characterization of CaCO₃ Crystals through Amperometry, FTIR, SEM-EDS, and XRD Analyses

Amperometrical curves, as with all pulsed techniques, chronoamperometry generates high charging currents at the beginning of the EC essays, which decay exponentially with time. Since the current is integrated over relatively longer time intervals, chronoamperometry gives a better signal to noise ratio in comparison to other amperometric techniques. Here, we observe a strong influence of the electrochemical current curve during the EC when Chi-P, as a solid template on the ITO substrate, was used (see Figure 2B). This behavior can be rationalized by increasing the amount of inorganic resistant material when the EC was performed at a constant voltage, then the applied current through the WE decayed until the stabilization of the crystallized CaCO₃ particles, which occurred in a very short time of 0.12 min at 0.17 mA. In this context, Chi-P induced the EC of CaCO₃ as a promoter effect, although, in both cases, the current became asymptotic until the end of the EC test was reached at 0.035 mA.

The FTIR results clearly demonstrated that although small amounts of inorganic materials were deposited onto the Chi-P film through the EC method, as supported by SEM analysis, all absorption bands of calcite polymorphism of CaCO₃ crystals can be identified by comparison with typical absorption bands of commercial CaCO₃ samples. As the Chi-P film could present a low surface chemical salt deposition, it is very likely that the resulting spectrum will mainly show bands corresponding to this derivative. However, the resulting CaCO₃ crystals’ bands (vaterite and calcite bands), as well as PO₄³⁻ salts, were clearly identified in the spectra from those of Chi-P, proving ATR/FTIR usefulness for material surface characterization (Figure 6 and Figure 7). In a complementary analysis, Raman spectroscopy was performed on the low amounts of CaCO₃ crystals grown on Chi-P surface, as was provided in supplementary material (please see Figure S4). Figure S4 shows the Raman spectra of ITO surface, starting Chi-P powder sample and CaCO₃ crystals as calcite polymorphism grown on Chi-P (Figure S4C) after EC experiments. The Raman spectra allowed us to also identify the deposited layer of CaCO₃ salt as calcite polymorphism confirming previous spectroscopic analysis.

In order to determine the elemental chemical information of all organic and inorganic components, that is, ITO substrate and the presence of Chi-P adsorbed on the crystal surface, energy dispersive X-ray spectroscopy (EDS) was also used. For the ITO substrate and Chi-P surface, the EDS measurements confirmed the presence of typical elemental composition of In, Sn, Al, and O for ITO (Figure 4A) and the C, O, and P for Chi-P (Figure 4B), respectively. The percentage of phosphorous content of the starting Chi-P sample was slightly higher of 4.0 wt% (Figure 4B). In Figure 4C, truncated crystals were found, suggesting an efficient modifier capacity of Chi-P during the EC of CaCO₃, although with a low concentration of phosphate groups on the film surface and low amounts of deposited crystals. EDS analysis determined a phosphorous content of 0.3 wt% of Chi-P immediately at the interface from CaCO₃ crystals and swollen Chi-P film (Figure 4D) (see Figure 4). Furthermore, EDS showed that theoretical calcium/oxygen ratio (Ca/O = 1/3) of the resultant CaCO₃ crystals in both flat and rounded analyzed area (symbol in the image) changed. In order to confirm these results in a broad area from spherical CaCO₃ particles, a flat and rounded area was selected and EDS was performed (Supplementary Materials Figure S5). This observation suggests that the adsorption of crystal particles onto Chi-P film does not occur preferentially on the Chi-P surface and the additive effect on the nucleation of CaCO₃ may occur due to the diffusion or more expanded (or soluble) chains of Chi-P, which can alter the process of crystal growth formation in order to favor a selective morphogenetic effect on CaCO₃. This effect could be due to the presence of small amount of phosphorylated Chi oligomers with higher water solubility than the original Chi-P polymer. Such oligomers could be solubilized and diffused from solid matrix to the aqueous media once the Chi-P film is swollen. It is known that Chi with phosphate derivatization in strong acid media may produce polymer degradation. With this in mind, SEM-EDS measurements of the resultant modified CaCO₃ particle growth onto the ITO surface in the presence of Chi-P, the Chi-P and ITO surface performed as we observed in Figure 4. On the other hand, similar truncate calcite and aggregated spherical particles of CaCO₃ were obtained when the GD method was performed. The morphological aspect of CaCO₃ crystals obtained in the absence and presence of Chi-P through the GD method and aggregated truncate calcite crystals are shown in Supplementary Material Figure S6.

Finally, SEM-EDS results are in qualitative agreement with all inorganic CaCO₃ particles obtained from both crystallization methods. It is important to mention that inspected CaCO₃ particles on the Chi-P surface were difficult to determine by spectroscopic analysis despite the large number of CaCO₃ particles layered on the Chi-P surface. Furthermore, an X-ray diffraction (XRD) analysis of the truncate calcite and spherical CaCO₃ particles obtained with the EC method was performed as shown in Figure 8A. Here, the XRD analysis clearly demonstrates the coexistence of calcite and vaterite polymorphs. It can be seen that the only two diffraction peaks appear at 2θ = 29.4° and at 2θ = 26.7°, which are ascribed to the most intense (104) and (112) crystallographic faces of calcite and vaterite, respectively. The diffraction peaks for calcite and vaterite for crystallographic planes and 2θ degree are in agreement with both crystals designations as was recently published using the ultrasound technique [59]. In the case of the XRD spectrum of Chi-P sample (Figure 8B), it shows a typical non-crystalline XRD curve, suggesting an amorphous nature of Chi-P biopolymer. The present data strongly suggest the ability of anionic functionalize Chi-P template for modifying the morphological aspect of CaCO₃ crystals and the stabilization capacity of the metastable form of vaterite. Consequently, the EC method offers a viable alternative for polymer-controlled crystallization of different inorganic minerals, such as Ca-salts, CaOx, hydroxyapatite, and silica, among others.

5. Conclusions

Phosphate moieties of Chi-P act showed inhibitor capability on the in vitro mineralization of CaCO₃ as we demonstrated in both crystallization methods and by using chronoamperometric curves of CaCO₃ obtained by the EC method. Since the phosphorous content of Chi-P was to be at ca. 3–4 wt% and CaCO₃ surface interaction is driven by anionic surface charge, we suggest that in vitro EC and GD methods are influenced by parameters such as current, voltage, phosphorous content, purity, pH, and topography surface of Chi-P, etc. The resultant CaCO₃ crystals obtained by EC method represents an advanced and fast in vitro mineralization system for studying different aspect of nucleation, crystal growth, size, and polymorphism of inorganic materials. For instance, Chi-P regulates the selectively precipitation of truncate and spherical aggregate CaCO₃ crystals in both mineralization systems. Here, ATR-FTIR and XRD analysis determined clearly the coexistence of calcite and the metastable form of vaterite as the unique polymorphism when EC method was performed. In summary, anionic Chi-P biopolymer acted as an efficient modifier of CaCO₃ crystallization using EC and GD methods and the EC demonstrated the viable capacity to control the polymorphism of CaCO₃. Therefore, the classical EC method offers a great chance for understanding the crucial role of functionalized biomacromolecules on the stabilization of metastable forms of crystals in the biomineralization field.