模型有机分子调控的碳酸钙仿生矿化研究
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摘要
生物矿物在形成过程中由于受到生物生长和生物大分子的调控作用而具有独特的形貌和复杂的组装超结构。本文分别以非离子三嵌段聚醚共聚物F68、含多羧基的柠檬酸钠和卵磷脂为矿化调节剂,通过仿生矿化方法,研究了这些模型矿化调节剂对碳酸钙矿物变体选择和形貌的影响。另外,通过选择合适的有机添加剂,合成了花状球霰石枝晶。其主要成果归纳如下:
1.利用三嵌段聚醚共聚物F68作为模型有机添加剂,通过仿生矿化方法,研究模型有机大分子对碳酸钙矿物结晶和生长过程的影响。结果显示,模型有机大分子F68不仅优先作用于方解石的某些特定晶面,使之形成拉长的微晶,而且诱导这些微晶沿着结晶学c轴方向取向聚集,形成方解石柱,即形成所谓的介晶结构方解石。此外,一系列时间过程矿化实验显示,在模型有机添加剂存在下,起始矿化产物是不稳定的非晶碳酸钙(Amorphous calcium carbonate, ACC);随着矿化时间的延长,这种暂时稳定的非晶前驱相通过一种介观尺度的转变(Mesoscale transformation)过程,最终转变成具有介晶结构的方解石柱,展现了与生物碳酸钙矿化过程相同的矿化序列和特征。尽管模型有机大分子F68仅含有大量醚氧基和端羟基官能团,但其在调控碳酸钙结晶过程中发挥的作用却类似于与生物矿化相关的糖基化蛋白等生物大分子。此外,以乙二醇替代F68的一系列控制实验结果显示,即使矿化体系中的乙二醇体积比高达20%,其矿化产物仍为菱面体方解石,指示着羟基对方解石特定形貌形成没有影响。因此,我们的实验结果可能暗示,在生物矿化过程中,除了极性羧基(COO-)外,与生物矿化相关的生物大分子糖蛋白中的非离子化的-C-O-C-基团(醚基或糖苷基)不仅影响着生物矿物的形成过程,而且对生物碳酸钙特定形貌的形成也有贡献。这些结果为更深刻地理解生物矿化的机制提供了新的途径。
2.选用含有多羧基的柠檬酸钠作为有机酸模型分子,进行了碳酸钙矿物的矿化模拟。结果表明,当柠檬酸钠浓度较低时,矿化体系中最初形成了纳米颗粒状含水的非晶碳酸钙(ACC),并暂时得到稳定,随后转化为棒状形貌的方解石,这种方解石棒最终演变成半球聚集体或球体。当柠檬酸钠浓度较高时,能够较长时间地稳定体系中形成的ACC,随着矿化时间的延长,ACC逐渐转变成由非晶核和棒形方解石壳构成的球。使用酒石酸钠替代柠檬酸钠进行的实验同样表明,较高浓度下,酒石酸钠在矿化的开始阶段同样诱导形成ACC。这些结果暗示着,在生物矿物形成过程中,与矿化相关的生物大分子中的一些极性基团如羧基可能也贡献了生物矿物非晶前驱体相的形成,并在非晶前驱体相后续转变成晶体矿物的过程中对矿物的特定形貌形成具有显著的影响。这些实验结果有助于深入理解与生物矿化过程紧密相关的酸性蛋白质在矿物形成过程中的调控作用。
3.利用卵磷脂作为影响矿物矿化过程的调节剂,在水溶液中仿生合成碳酸钙。结果表明,卵磷脂可以诱导形成不常见的无水ACC,并影响这种ACC后续转变形成的方解石的形貌。当溶液中卵磷脂浓度较低时,不足以稳定溶液中形成的ACC,以致于没有观察到ACC的存在;但卵磷脂形成的胶束利用极性磷酸酯基作用于形成的方解石的晶体表面,使之形成多孔的形貌。而当卵磷脂浓度较高时,卵磷脂在溶液中能够诱导形成并暂时稳定不含结构水的ACC;且在ACC后续转变为方解石的过程中影响着方解石的形貌。这些结果指示,卵磷脂对碳酸钙非晶前驱体相的形成和后续转变形成的矿物形貌产生了明显的影响。由于大部分生物矿物都经历了由无定形态前驱体到结晶相的转变过程,卵磷脂在合适的浓度条件下可以诱导形成并暂时稳定无水ACC,并影响后续转变而成的碳酸钙晶体的形貌,这一点对我们理解生物体内不常见的无水ACC和特殊形貌的矿物的形成过程具有重要的意义。
4.利用微波快速加热的方式合成了花状的球霰石枝晶。球霰石是碳酸钙矿物的热力学不稳定相,其形成过程往往受动力学因素控制。最近有关生物碳酸钙矿化研究显示,许多生物过程能产生稳定的球霰石,这就指示生物大分子可能稳定不稳定球霰石;此外,微波快速加热能使被加热物质的温度在很短的时间内迅速升高且没有温度梯度,往往有利于亚稳相,即动力学控制相的形成。基于这样思想,我们通过微波快速加热和有机添加剂协调作用过程,成功地制备了纯的花状球霰石枝晶。实验结果显示,在以去离子水为溶剂时,得到了菱面体状方解石;但在反应体系中加入少量的乙二醇后得到了方解石和少量扁平盘状球霰石的混合相。在体系中加入2-萘基氧乙酸作为有机添加剂,尽管2-萘基氧乙酸有助于形成球霰石,但由于其在水中的溶解性较差,也获得了方解石和球霰石的混合相。而在体系中加入少量的乙二醇溶解2-萘基氧乙酸后,获得了纯的花状球霰石枝晶。在球霰石的形成过程中,2-萘基氧乙酸利用羧基的负电性有效地中和了其高能(001)面的电荷,降低了该晶面的能量,使得球霰石沿着平行于该晶面的方向生长,形成了花状球霰石枝晶。实验成功合成了花状形貌的球霰石枝晶,同时对这种花状形貌晶体形成机制进行了探讨。
Biogenetic minerals possess unique morphologies and complex architectures due to the precise regulation in the mineralization by the biomacromolecules in vivo and the growth of organisms. In this dissertation, nonionic pluronic triblock copolymer of F68, carboxylate-enriched sodium citrate and phosphatidylcholine(PC) were respectively served as model mineralized modifiers to study the effects of these modifiers on the polymorph selection and the formation of morphology of calcium carbonate via a biomimetic mineralization method. In addition, flower-shaped vaterite dendrites were successfully prepared by selecting suitable organic additives as the modifiers. Some important results were summarized as follows:
1. A pluronic triblock copolymer of F68 was selected as model organic additive to influence the crystallization and growth of calcium carbonate by a biomimetic mineralization process. The results demonstrated that the model organic macromolecule of F68 not only preferentially interacted with the particular faces of calcite crystals to form elongated microcrystals, but also induced the oriented aggregation of these microcrystals along the crystallographic c direction into calcite prisms, i.e., forming mesocrystal architectures. In addition, a series of time-resolved experiments showed that the initial precipitate in the presence of higher concentration of F68 was unstable amorphous calcium carbonate(ACC), and this transient precursor phase eventually transformed into prismatic mesocrystals of calcite via a mesoscale transformation process, which displays the same biomineralized sequence and features with the biomineralized process of biogenetic CaCO3. Although F68 only contains mass of ether-oxygen groups and a terminal hydroxyl group, its role played in the regulation of calcium carbonate crystallization process is considered analogous to an array of biomineralization-associated biomacromolecules such as some glycosylated proteins. Furthermore, the results of a series of control experiments via the replacement of F68 by ethylene glycol displayed that the minerallized products were calcitic rhombohedra even though the volume ratio of ethylene glycol was increased to 20% in the mineralized system. This indicated that the hydroxyl groups had no effect on the formation of the particular morphology of calcite. Hence, our experimental results suggest that apart from the polar carboxyl, nonionic -C-O-C- group (ether or glycosidic group) in the biomineralization-associated biomacromolecules of glycosidoproteins may not only influence the formation process but also contribute to the special morphogenesis of the biominerals. As such, these results provide another pathway towards a deeper insight into biomineralization mechanism.
2. Multi-carboxylic sodium citrate was applied as the model acidic molecule to influence the mineralization of calcium carbonate. The results showed that with a low concentration of sodium citrate in the mineralized system, ACC nanoparticles were first formed and temporarily stabilized in the mineralized system, and then these ACC nanoparticles transformed into calcite with rod-like morphology. These calcite rods aggregated and finally evolved into hemispheres or spheres. With a high concentration, sodium citrate could stabilize the ACC nanoparticles for a long time and simultaneously made ACC nanoparticles assembly into spherical particles. In the following mineralization process, ACC nanoparticles gradually transformed into spheres with nuclear-shell structures, and the nuclei were composed of ACC nanoparticles while slim calcitic rods constituted the shells. The similar experiment by using of sodium tartrate to replace sodium citrate also show that sodium tartrate with a high concentration was capble of inducing the formation of ACC in the initial stage of mineralization as well. These results indicate that in biomineral formation process, some polar groups such as carboxyls in the biomineralization-associated biomacromolecules may not only contribute to the formation of amorphous precursor phases of the biominerals but also to the influence of the morphogenesis of the crystalline biominerals which transform from the amorphous precursor phases. These results provide us a deeper insight into the significances of the acidic proteins in vivo in the regulation of the formation process of biominerals.
3. By use of phosphatidylcholine(PC) as the modifier of mineralization process, CaCO_3 was biomimetically synthesized in aqueous solution. The results showed that PC is capable of inducing the formation of the unusal anhydrous ACC and influencing the morphology of calcite which tansformed from ACC in the follow-up mineralization process. With a lower concentration of PC in solution, PC was insufficient to stabilize transient ACC which formed in the initial stage of mineralization, therefore, ACC was not detected in the as-obtained products. However, PC was capable of making calcitic crystals to form porous surface morphologies by the interaction between the polar phosphoric ester groups and the surfaces of calcite. With a higher concentration of PC in solution, PC can induce the formation of ACC and temporarily stablize this transient phase of calcium carbonate using the negatively charged phosphoric ester groups and affect the morphology of the mineral in the follow-up process of ACC transformed into calcite.These results indicate that PC is able to induce the formation of transient anhydrous ACC and influence the morphology of calcite which from the transformation of ACC. Since the great mass of biominerals undergo phase transitions from amorphous precursors into crystalline states, PC can stabilize ACC with certain concentration and affect the morphogenesis of CaCO_3
4. Flower-shaped vaterite dendrites were successfully synthesized by a rapid microwave-assisted heating method. Vaterite is a thermodynamically unstable phase of calcium carbonate, so its formation is often controlled by kinetic factors. Recent studies of the mineralization of biogenetic calcium carbonate show that a large number of biological processes are capable of forming stable vaterite, which may indicate that the biological macromolecules can stabilize metastable vaterite. In addition, rapid microwave heating enables the temperature of the heated material to increase rapidly in a very short period of time without temperature gradient, which tends to favor the formation of the metastable phase, namely the kinetics control phase. Based on these thoughts, we successfully prepared pure flower-shaped vaterite dendrites through the coordinating process of by rapid microwave heating and suitable organic additives. The results showed that the rhombohedral calcite was the exclusive product when deionized water was applyed as the solvent; however, rhombohedral calcite and a spot of vaterite with planular discoid morphology were obtained by adding a small amount of ethylene glycol in the reaction system. With the introduction of 2-naphthyl oxygen acetic acid as an organic additive which contributes to the formation of metastable vaterite into the reaction system, the mixed phases which were composed of rhombohedral calcite, ball and flower-shaped vaterite were obtained in aqueous solution because of 2-naphthyl oxygen acetic acid poor solubility in water. However, with adding a small amount of ethylene glycol into the reaction system to resolve 2-naphthyl oxygen acetic acid, flower-shaped vaterite dendrites were obtained. In the formation process of metastable vaterite, 2-naphthyl oxygen acetic acid can apply the electronegativity of the polar carboxyl to effectively neutralize the charge of the high-energy (001) surface of vaterite and reduce the crystal surface energy. This results in the vaterite growth oriented along parallel to the , which is of great importance for the comprehension of the formation process of ACC and calcium carbonate with special morphologies in vivo. (001) plane and the formation of flower-shaped vaterite dendrites. Flower-like vaterite dendrites were successfully synthesized in this work and the formation mechanism of these dentrites were investigated as well.
1.利用三嵌段聚醚共聚物F68作为模型有机添加剂,通过仿生矿化方法,研究模型有机大分子对碳酸钙矿物结晶和生长过程的影响。结果显示,模型有机大分子F68不仅优先作用于方解石的某些特定晶面,使之形成拉长的微晶,而且诱导这些微晶沿着结晶学c轴方向取向聚集,形成方解石柱,即形成所谓的介晶结构方解石。此外,一系列时间过程矿化实验显示,在模型有机添加剂存在下,起始矿化产物是不稳定的非晶碳酸钙(Amorphous calcium carbonate, ACC);随着矿化时间的延长,这种暂时稳定的非晶前驱相通过一种介观尺度的转变(Mesoscale transformation)过程,最终转变成具有介晶结构的方解石柱,展现了与生物碳酸钙矿化过程相同的矿化序列和特征。尽管模型有机大分子F68仅含有大量醚氧基和端羟基官能团,但其在调控碳酸钙结晶过程中发挥的作用却类似于与生物矿化相关的糖基化蛋白等生物大分子。此外,以乙二醇替代F68的一系列控制实验结果显示,即使矿化体系中的乙二醇体积比高达20%,其矿化产物仍为菱面体方解石,指示着羟基对方解石特定形貌形成没有影响。因此,我们的实验结果可能暗示,在生物矿化过程中,除了极性羧基(COO-)外,与生物矿化相关的生物大分子糖蛋白中的非离子化的-C-O-C-基团(醚基或糖苷基)不仅影响着生物矿物的形成过程,而且对生物碳酸钙特定形貌的形成也有贡献。这些结果为更深刻地理解生物矿化的机制提供了新的途径。
2.选用含有多羧基的柠檬酸钠作为有机酸模型分子,进行了碳酸钙矿物的矿化模拟。结果表明,当柠檬酸钠浓度较低时,矿化体系中最初形成了纳米颗粒状含水的非晶碳酸钙(ACC),并暂时得到稳定,随后转化为棒状形貌的方解石,这种方解石棒最终演变成半球聚集体或球体。当柠檬酸钠浓度较高时,能够较长时间地稳定体系中形成的ACC,随着矿化时间的延长,ACC逐渐转变成由非晶核和棒形方解石壳构成的球。使用酒石酸钠替代柠檬酸钠进行的实验同样表明,较高浓度下,酒石酸钠在矿化的开始阶段同样诱导形成ACC。这些结果暗示着,在生物矿物形成过程中,与矿化相关的生物大分子中的一些极性基团如羧基可能也贡献了生物矿物非晶前驱体相的形成,并在非晶前驱体相后续转变成晶体矿物的过程中对矿物的特定形貌形成具有显著的影响。这些实验结果有助于深入理解与生物矿化过程紧密相关的酸性蛋白质在矿物形成过程中的调控作用。
3.利用卵磷脂作为影响矿物矿化过程的调节剂,在水溶液中仿生合成碳酸钙。结果表明,卵磷脂可以诱导形成不常见的无水ACC,并影响这种ACC后续转变形成的方解石的形貌。当溶液中卵磷脂浓度较低时,不足以稳定溶液中形成的ACC,以致于没有观察到ACC的存在;但卵磷脂形成的胶束利用极性磷酸酯基作用于形成的方解石的晶体表面,使之形成多孔的形貌。而当卵磷脂浓度较高时,卵磷脂在溶液中能够诱导形成并暂时稳定不含结构水的ACC;且在ACC后续转变为方解石的过程中影响着方解石的形貌。这些结果指示,卵磷脂对碳酸钙非晶前驱体相的形成和后续转变形成的矿物形貌产生了明显的影响。由于大部分生物矿物都经历了由无定形态前驱体到结晶相的转变过程,卵磷脂在合适的浓度条件下可以诱导形成并暂时稳定无水ACC,并影响后续转变而成的碳酸钙晶体的形貌,这一点对我们理解生物体内不常见的无水ACC和特殊形貌的矿物的形成过程具有重要的意义。
4.利用微波快速加热的方式合成了花状的球霰石枝晶。球霰石是碳酸钙矿物的热力学不稳定相,其形成过程往往受动力学因素控制。最近有关生物碳酸钙矿化研究显示,许多生物过程能产生稳定的球霰石,这就指示生物大分子可能稳定不稳定球霰石;此外,微波快速加热能使被加热物质的温度在很短的时间内迅速升高且没有温度梯度,往往有利于亚稳相,即动力学控制相的形成。基于这样思想,我们通过微波快速加热和有机添加剂协调作用过程,成功地制备了纯的花状球霰石枝晶。实验结果显示,在以去离子水为溶剂时,得到了菱面体状方解石;但在反应体系中加入少量的乙二醇后得到了方解石和少量扁平盘状球霰石的混合相。在体系中加入2-萘基氧乙酸作为有机添加剂,尽管2-萘基氧乙酸有助于形成球霰石,但由于其在水中的溶解性较差,也获得了方解石和球霰石的混合相。而在体系中加入少量的乙二醇溶解2-萘基氧乙酸后,获得了纯的花状球霰石枝晶。在球霰石的形成过程中,2-萘基氧乙酸利用羧基的负电性有效地中和了其高能(001)面的电荷,降低了该晶面的能量,使得球霰石沿着平行于该晶面的方向生长,形成了花状球霰石枝晶。实验成功合成了花状形貌的球霰石枝晶,同时对这种花状形貌晶体形成机制进行了探讨。
Biogenetic minerals possess unique morphologies and complex architectures due to the precise regulation in the mineralization by the biomacromolecules in vivo and the growth of organisms. In this dissertation, nonionic pluronic triblock copolymer of F68, carboxylate-enriched sodium citrate and phosphatidylcholine(PC) were respectively served as model mineralized modifiers to study the effects of these modifiers on the polymorph selection and the formation of morphology of calcium carbonate via a biomimetic mineralization method. In addition, flower-shaped vaterite dendrites were successfully prepared by selecting suitable organic additives as the modifiers. Some important results were summarized as follows:
1. A pluronic triblock copolymer of F68 was selected as model organic additive to influence the crystallization and growth of calcium carbonate by a biomimetic mineralization process. The results demonstrated that the model organic macromolecule of F68 not only preferentially interacted with the particular faces of calcite crystals to form elongated microcrystals, but also induced the oriented aggregation of these microcrystals along the crystallographic c direction into calcite prisms, i.e., forming mesocrystal architectures. In addition, a series of time-resolved experiments showed that the initial precipitate in the presence of higher concentration of F68 was unstable amorphous calcium carbonate(ACC), and this transient precursor phase eventually transformed into prismatic mesocrystals of calcite via a mesoscale transformation process, which displays the same biomineralized sequence and features with the biomineralized process of biogenetic CaCO3. Although F68 only contains mass of ether-oxygen groups and a terminal hydroxyl group, its role played in the regulation of calcium carbonate crystallization process is considered analogous to an array of biomineralization-associated biomacromolecules such as some glycosylated proteins. Furthermore, the results of a series of control experiments via the replacement of F68 by ethylene glycol displayed that the minerallized products were calcitic rhombohedra even though the volume ratio of ethylene glycol was increased to 20% in the mineralized system. This indicated that the hydroxyl groups had no effect on the formation of the particular morphology of calcite. Hence, our experimental results suggest that apart from the polar carboxyl, nonionic -C-O-C- group (ether or glycosidic group) in the biomineralization-associated biomacromolecules of glycosidoproteins may not only influence the formation process but also contribute to the special morphogenesis of the biominerals. As such, these results provide another pathway towards a deeper insight into biomineralization mechanism.
2. Multi-carboxylic sodium citrate was applied as the model acidic molecule to influence the mineralization of calcium carbonate. The results showed that with a low concentration of sodium citrate in the mineralized system, ACC nanoparticles were first formed and temporarily stabilized in the mineralized system, and then these ACC nanoparticles transformed into calcite with rod-like morphology. These calcite rods aggregated and finally evolved into hemispheres or spheres. With a high concentration, sodium citrate could stabilize the ACC nanoparticles for a long time and simultaneously made ACC nanoparticles assembly into spherical particles. In the following mineralization process, ACC nanoparticles gradually transformed into spheres with nuclear-shell structures, and the nuclei were composed of ACC nanoparticles while slim calcitic rods constituted the shells. The similar experiment by using of sodium tartrate to replace sodium citrate also show that sodium tartrate with a high concentration was capble of inducing the formation of ACC in the initial stage of mineralization as well. These results indicate that in biomineral formation process, some polar groups such as carboxyls in the biomineralization-associated biomacromolecules may not only contribute to the formation of amorphous precursor phases of the biominerals but also to the influence of the morphogenesis of the crystalline biominerals which transform from the amorphous precursor phases. These results provide us a deeper insight into the significances of the acidic proteins in vivo in the regulation of the formation process of biominerals.
3. By use of phosphatidylcholine(PC) as the modifier of mineralization process, CaCO_3 was biomimetically synthesized in aqueous solution. The results showed that PC is capable of inducing the formation of the unusal anhydrous ACC and influencing the morphology of calcite which tansformed from ACC in the follow-up mineralization process. With a lower concentration of PC in solution, PC was insufficient to stabilize transient ACC which formed in the initial stage of mineralization, therefore, ACC was not detected in the as-obtained products. However, PC was capable of making calcitic crystals to form porous surface morphologies by the interaction between the polar phosphoric ester groups and the surfaces of calcite. With a higher concentration of PC in solution, PC can induce the formation of ACC and temporarily stablize this transient phase of calcium carbonate using the negatively charged phosphoric ester groups and affect the morphology of the mineral in the follow-up process of ACC transformed into calcite.These results indicate that PC is able to induce the formation of transient anhydrous ACC and influence the morphology of calcite which from the transformation of ACC. Since the great mass of biominerals undergo phase transitions from amorphous precursors into crystalline states, PC can stabilize ACC with certain concentration and affect the morphogenesis of CaCO_3
4. Flower-shaped vaterite dendrites were successfully synthesized by a rapid microwave-assisted heating method. Vaterite is a thermodynamically unstable phase of calcium carbonate, so its formation is often controlled by kinetic factors. Recent studies of the mineralization of biogenetic calcium carbonate show that a large number of biological processes are capable of forming stable vaterite, which may indicate that the biological macromolecules can stabilize metastable vaterite. In addition, rapid microwave heating enables the temperature of the heated material to increase rapidly in a very short period of time without temperature gradient, which tends to favor the formation of the metastable phase, namely the kinetics control phase. Based on these thoughts, we successfully prepared pure flower-shaped vaterite dendrites through the coordinating process of by rapid microwave heating and suitable organic additives. The results showed that the rhombohedral calcite was the exclusive product when deionized water was applyed as the solvent; however, rhombohedral calcite and a spot of vaterite with planular discoid morphology were obtained by adding a small amount of ethylene glycol in the reaction system. With the introduction of 2-naphthyl oxygen acetic acid as an organic additive which contributes to the formation of metastable vaterite into the reaction system, the mixed phases which were composed of rhombohedral calcite, ball and flower-shaped vaterite were obtained in aqueous solution because of 2-naphthyl oxygen acetic acid poor solubility in water. However, with adding a small amount of ethylene glycol into the reaction system to resolve 2-naphthyl oxygen acetic acid, flower-shaped vaterite dendrites were obtained. In the formation process of metastable vaterite, 2-naphthyl oxygen acetic acid can apply the electronegativity of the polar carboxyl to effectively neutralize the charge of the high-energy (001) surface of vaterite and reduce the crystal surface energy. This results in the vaterite growth oriented along parallel to the , which is of great importance for the comprehension of the formation process of ACC and calcium carbonate with special morphologies in vivo. (001) plane and the formation of flower-shaped vaterite dendrites. Flower-like vaterite dendrites were successfully synthesized in this work and the formation mechanism of these dentrites were investigated as well.
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