Biomineralization is the process of inorganic crystal growth at inorganic-organic interface    , which exerts a significant influence on the growth of life. Therefore, the researches about the biomineralization have aroused many interests  -  . From the number of carbonate minerals, CaCO3 is the most important biominerals in various industrial fields, such as additives in medicine, foods, plastics, printing ink, cosmetic, etc. Its final application is determined by its purity, particle size and distribution, crystal structure and morphology  . Some studies indicated that high-density Mg2+ was found in bio-environment and it took an important part in the biomineralization of calcium carbonate   . Eva Loste et al. have studied the role of magnesium in stabilizing amorphous calcium carbonate and controlling calcite morphologies  . Yong-Jin Han et al. have investigated effect of Mg2+ on oriented growth of calcite on carboxylic acid functionalized self-assembled monolayers  . Davis et al. have indicated that the role of Mg2+ as an impurity in calcite growth  . NorioWada et al. have demonstrated the effects of divalent cations upon nucleation, growth and transformation of calcium carbonate polymorphs under conditions of double diffusion  . Yun-Feng Jiao et al. studied the co-effect of collagen and magnesium ions on calcium carbonate biomineralization  . An-jian Xie et al. investigate the role of Mg2+ and Mg2+/amino acid in controlling polymorph and morphology of calcium carbonate crystal  . Li-Ying Zhu et al. synthesize star shaped calcite with Mg2+ without organic template in the mixed solvent of ethanol and H2O  . However, the effect of different concentrations Mg2+ on the polymorph and morphology of CaCO3 crystal using protein Langmuir monolayers method was not described systemically so far. Further, protein as an organic matrix also plays a critical role in the biomineralization of the CaCO3   , it exists at inorganic-organic interface of almost all the biominerals such as teeth, bones, pearls, mollusk shells   . They may be important factors in controlling of CaCO3 polymorphs and morphologies. Many studies on the combined effect of organic additives and magnesium ions on calcium carbonate crystal growth have been reported recently    , but little research has been carried out on the cooperative influence of protein Langmuir monolayers and magnesium ions on calcium carbonate precipitation.
This paper mainly discusses the cooperative influence of BSA Langmuir monolayers and magnesium ions on calcium carbonate precipitation. The aim of these experiments is to find the effect on the polymorph and morphology of calcium carbonate by combined BSA Langmuir monolayers and magnesium ions. Simultaneously, the possible formation mechanism of CaCO3 crystals on the basis of the principle of biomineralization was discussed. It is significant to understand the mechanism of biomineralization and synthesizing new and special functional materials.
2. Experimental Section
BSA was purchased from Sigma and used without further purification. MgCl2∙6H2O, CaCO3 and Amyl alcohol were all analytical purity. All the aqueous solutions in our experiment were prepared by using the triply deionized water, its conductivity was a resistance of 18.2 MΩ・cm−1 and its pH was 7.0.
2.2. Crystallization of CaCO3
Supersaturated solutions of calcium bicarbonate were prepared according to the procedures of Kitano  . MgCl2・6H2O was dissolved into Ca(HCO3)2 aqueous solution(5 mmolL−1) and the pH value of solution was 7.0. The spreading monolayers at the air-water interface were formed by spreading solutions of BSA (1 × 10−4 mol∙L−1) on the pure water surface or Supersaturated solutions of calcium bicarbonate containing Mg2+. A 30-min lapse time was estimated to be sufficient to equilibrate the protein monolayers before compression. We used very low compression rates and choose 15 mN/m as a target surface pressure, which has been shown to be appropriate to obtain reproducible isotherms and stable BSA Langmuir monolayers  . In the different concentration of Mg2+ system, Mg2+/Ca2+ molar ratio was 1:1; 2:1; 3:1; 4:1; 5:1 respectively. The prepared supersaturated calcium bicarbonate solutions were poured into a Langmuir trough and the air-water interface was swept and aspirated before deposition of the surfactant solution. The surfactant solution (10 µL) was carefully deposited onto the solution surface. Each experiment was repeated three times with the same condition. Crystals as-grown in association with the monolayers were respectively removed after 4 h by carefully horizontally dipping hydrophilic glass slides through the air-water interface. The crystal face growing into the solution is therefore directly deposited on the glass slide.
2.3. Characterization of Crystal Phase and Morphology of CaCO3
The sizes and morphologies of CaCO3 were characterized by using SEM on JSM-5600LV scanning electron microscopy (Jeol. Ltd. Japan) with operating at 30 kV. The slides supporting the crystals were mounted on copper sample stubs with conducting carbon tape and were sputter-coated with gold prior to viewing. The XRD measurements were performed by a (Philips X’Pert Pro) X-ray powder diffractometer using a monochromatized Cu Kα1 radiation (λ = 1.5406 Å).
3. Results and Discussion
3.1. The Influence of BSA Langmuir Monolayers and Magnesium on Polymorph of CaCO3
From XRD it can be seen that the polymorph of CaCO3, obtained at the interface of film/solution changed with increasing the concentration of magnesium in the solution. When the ratio of Mg/Ca is 1 only calcite crystals are formed (Figure 1(a)), and when the ratio of Mg/Ca is 2 some vaterite and aragonite crystals are precipitated besides calcite (Figure 1(b)). And when the ratio of Mg/Ca attain 3 almost the crystals are aragonite, only a few are calcite crystals (Figure 1(c)). While all crystals are aragonite when the ratio of Mg/Ca is 4 and 5 (Figure 1(d),
Figure 1. XRD patterns of CaCO3 particles obtained from different Mg2+/Ca2+ molar ratio systems. (a) 1:1; (b) 2:1; (c) 3:1; (d) 4:1; (e) 5:1. (f) without Mg2+ in solutions.
Figure 1(e)). A lot of reports indicate that only at ratio of Mg/Ca of 4 or above that aragonite can be seen if there is no organic additive or biopolymer present, in other instance only calcite can be seen, which is in agreement with conventional theoretical results. When BSA Langmuir monolayers are present alone, only calcite crystals are formed (Figure 1(f)). Magnesium is known to induce aragonite formation from sea water and in vitro at ratio of Mg/Ca equal to or greater than 4, while at lower Mg/Ca ratio mostly calcite and magnesian calcite are formed  . The above results show that when BSA Langmuir monolayers and magnesium are both present, aragonite crystals precipitated at a lower Mg/Ca ratio of 2. This indicates that BSA Langmuir monolayers have a promotional effect on magnesium ions in controlling the polymorph of CaCO3 crystals.
3.2. The Influence of BSA Langmuir Monolayers and Magnesium on Morphology of CaCO3
A much wide range of calcium carbonate morphologies is generated in the presence of both BSA Langmuir monolayers and magnesium ions. At low magnesium concentration (Mg/Ca ratio is 1), regular abacus-bead-like calcite crystals are formed (Figure 2(a)). When the concentration of magnesium is high enough (Mg/Ca ratio attains to 2 and 3), wood-block-like aragonite crystals are precipitated (Figure 2(b), Figure 2(c)). In order to further study the morphology
Figure 2. SEM images of CaCO3 particles obtained from different Mg2+/Ca2+ molar ratio systems. (a) 1:1; (b) 2:1; (c) 3:1; (d) 4:1; (e) 5:1. (f) without Mg2+ in solutions.
evolvement of calcium carbonate, the higher Mg2+/Ca2+ ratio (4:1 and 5:1) was observed. The results indicated that the pignut-shell-like and rolling pole shaped aragonite crystals were produced at ratio of Mg2+/Ca2+ 4:1 and 5:1, respectively (Figure 2(d), Figure 2(e)). Figure 2(f) is the SEM image of calcite obtained without Mg2+, it shows that the calcite took jujube-nucleus-like morphology, which is different from the results obtained at solution containing Mg2+.
Above results showed that in the presence of BSA Langmuir monolayers, when magnesium is added, the morphology of crystals changed greatly, from abacus-bead to wood-block, pignut-shell and rolling pole. The morphological changes of calcium carbonate crystals reveal that in the presence of both BSA Langmuir monolayers and magnesium ions, we tend to obtain various morphological aragonite. Especially when the concentration of magnesium is high, the aragonite crystals with more regular shapes could be obtained.
The reason of this kinetic phenomenon is considered to contribute to two aspects: on the one hand, magnesium ions inhibit the growth of calcite. The partially dehydrated magnesium ions attach to the surface of the nascent calcite nucleus, the strongly bound residual hydration sphere poisons the surface  , and inhibits subsequent growth. At the same time, the BSA acts with magnesium and increases the magnesium hydrate absorbed in calcite nucleus. On the other hand, owing to the polymorph of calcium carbonate is relate to its energy state  , magnesium is likely also to react with BSA and to change the secondary structure of BSA molecules, and thus induces the aragonite or vaterite with higher energy in high energy state structure.
According to the nucleation and growth theory    , to form a new nucleus, the activation energy (ΔGN) must be overcome. ΔGN can be expressed as:
where ΔG1 is the surface energy that was needed to form the new interface and maintain the crystal growth, k is the Boltzmann constant, T is the temperature, and S is the supersaturation of area. The decrease of the surface energy ΔG1 or the increase of the S can reduce the activation energy for crystal nucleation. The isoelectric points (pI = 4.7) of BSA were both lower than the pH (7.0) of aqueous solution, the negative charges on the surface of BSA were surplus, they could attract Ca2+ strongly because of electrostatic interactions, it aroused the concentration of Ca2+ was rich in this region. At the same time, the present of magnesium ions during the calcium carbonate precipitation in vitro in known to inhibit the growth of calcium carbonate and consequently increase the degree of supersaturation  , such conditions enable the formation of metastable phases in general  ; meanwhile BSA that offered nucleation sites for the growth of CaCO3 particles could reduce the surface energy, so ΔGN declined, which was helpful for the formation of the high-energy aragonite. From these results we could see that the molar ratio of Mg2+/Ca2+ has very important effect on the morphology of CaCO3. However, the precise role of magnesium ions in the stabilization of the metastable phases remains enigmatic.
The present paper mainly studied the cooperative effect of BSA Langmuir monolayers and magnesium ions on polymorph and morphology of calcium carbonate crystal. The results indicated that BSA Langmuir monolayers and magnesium ions have a promotional effect on magnesium ions in controlling the polymorph of CaCO3 crystals. By cooperation of BSA Langmuir monolayers and magnesium ions in different concentrations, crystals showed a sequence of morphology changes and especially, aragonite crystals with various unusual morphology were precipitated at high Mg2+/Ca2+ ions concentration ratio. The experiments suggested that BSA Langmuir monolayers act in combination with magnesium ions to inhibit calcite crystal growth, while favors the formation of aragonite crystals. It indicated that both Mg2+ and proteins played an essential importance role in the process of biomineralization of CaCO3.
This work was supported by the National Natural Science Foundation of China, Grant No. 21203055.
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