Nanospheres
ATP-Stabilized Amorphous Calcium Carbonate Nanospheres and Their Application in Protein Adsorption Chao Qi, Ying-Jie Zhu,* Bing-Qiang Lu, Xin-Yu Zhao, Jing Zhao, Feng Chen, and Jin Wu
Calcium carbonate is a common substance found in rocks worldwide, and is the main biomineral formed in shells of marine organisms and snails, pearls and eggshells. Amorphous calcium carbonate (ACC) is the least stable polymorph of calcium carbonate, which is so unstable under normal conditions that it is difficult to be prepared in vitro because it rapidly crystallizes to form one of the more stable polymorphs in aqueous solution. Herein, we report the successful synthesis of highly stable ACC nanospheres in vitro using adenosine 5′-triphosphate disodium salt (ATP) as a stabilizer. The effect of ATP on the stability of ACC nanospheres is investigated. Our experiments show that ATP plays an unique role in the stabilization of ACC nanospheres in aqueous solution. Moreover, the as-prepared ACC nanospheres are highly stable in phosphate buffered saline for a relatively long period of time (12 days) even under relatively high concentrations of calcium and phosphate ions. The cytotoxicity tests show that the as-prepared highly stable ACC nanospheres have excellent biocompatibility. The highly stable ACC nanospheres have high protein adsorption capacity, implying that they are promising for applications in biomedical fields such as drug delivery and protein adsorption.
1. Introduction Calcium carbonate (CaCO3), an ubiquitous and important biomineral in biological systems,[1,2] has aroused enormous interest because it is widely used as a model system for studying the biomimetic processes that are very common and important phenomena in nature.[3–6] Organisms have the ability of biomineralization to form a composite material composed of calcium carbonate embedded in the organic matrix,[7–9] which inspires researchers to synthesize calcium C. Qi, Prof. Dr. Y.-J. Zhu, Dr. B.-Q. Lu, X.-Y. Zhao, J. Zhao, F. Chen, Dr. J. Wu State Key Laboratory of High Performance Ceramics and Superfine Microstructure Shanghai Institute of Ceramics Chinese Academy of Sciences Shanghai 200050, P. R. China Tel: 0086-21-52412616; Fax: 0086-21-52413122 E-mail: [email protected] DOI: 10.1002/smll.201302984 small 2014, 10, No. 10, 2047–2056
carbonate in vitro. Calcium carbonate occurs naturally in three anhydrous crystalline polymorphs (calcite, aragonite and vaterite in the order of decreasing stability), two hydrated metastable forms (monohydrocalcite and calcium carbonate hexahydrate), and an unstable amorphous calcium carbonate (ACC) phase.[10–12] Among these polymorphs, ACC is the most important phase because it plays a pivotal role in the process of biomineralization for other phases and is the precursor to the crystalline polymorphs.[13,14] ACC has several potential biological functions due to its high solubility relative to the crystalline phases. On the other hand, the bioavailability of ACC, commonly used medicinally as a calcium supplement or as an antacid, is roughly 40% more bioavailable than the crystalline calcium carbonate with only around 20–30%.[15] Due to lower stability and higher solubility of ACC compared with the crystalline phases of calcium carbonate, it is difficult to produce pure ACC in vitro, and ACC will crystallize rapidly to form one of the more stable polymorphs in aqueous solution.[16] However, the highly stable ACC, stabilized by specific biological macromolecules which is a
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C. Qi et al.
key principle in the nature of biomineralization,[17–20] has been found to exist in living organisms such as in ascidian skeleton, plant cystoliths, spicules from sponge and mollusk shells.[21,22] Inspired by biomineralization, many researchers have devoted to the design and synthesis of stabilized ACC with some additives such as biomolecules and organic molecules under mild conditions in vitro.[23–26] For example, Huang et al.[27] managed to stabilize ACC for several months using polyacrylic acid. In addition, the inorganic ions such as magnesium ions, phosphates as well as silica are also capable of stabilizing ACC for a certain period.[28–32] Freeze-drying can also produce stable ACC.[33] Bentov et al.[34] reported that phosphorylated amino acids could produce ACC with long-term stability. However, only a few methods have been reported for the synthesis of ACC which is stable in aqueous solution for more than several weeks. In order to understand the biomineralization mechanism of calcium carbonate, it is necessary to explore the effects of biomolecules on biomineralization. Adenosine 5′-triphosphate (ATP) is the most common energy carrier of the cell in the biological system, and plays an important role in various life activities for transporting chemical energy within cells for metabolism. Previous works indicate that ATP can be used as a stabilizer for amorphous calcium phosphate to inhibit the crystallization process in aqueous solution by poisoning heterogeneous nucleation sites and binding to embryonic crystal nucleus.[35–37] Moreover, ATP can strongly inhibit the formation of calcium carbonate crystals and induce deposition of amorphous calcium carbonate.[38] Herein, we report the successful synthesis of highly stable ACC nanospheres in vitro using ATP as the stabilizer (Scheme 1). The effect of ATP biomolecules on the stability of ACC nanospheres in aqueous solution is investigated. Moreover, the as-prepared ACC nanospheres are highly stable in phosphate buffered saline (PBS) for a relatively long period of time (12 days) even at relatively high concentrations of calcium ions and phosphate ions, which is important for the investigation of the biomineralization processes. Furthermore, the
Table 1. Experimental conditions for the preparation of typical samples using aqueous solutions containing CaCl2, Na2CO3 and ATP (pH 9) and the ATP contents of the products. Sample No.
ATP concentration [mM]
Stirring time at room temp. [h]
ATP content [wt.%]
1
–
1
–
2
1
1
18.373
3
2.5
1
26.738
4
4
1
37.561
5
5
1
49.565
6
5
120
43.589
cytotoxicity tests show that the as-prepared highly stable ACC nanospheres have excellent biocompatibility. The highly stable ACC nanospheres can be used as the excellent adsorbent with high protein adsorption capacity, indicating that they are promising for applications in biomedical fields such as drug delivery and protein adsorption.
2. Results and Discussion 2.1. Formation and Characterization of ACC Nanospheres Stabilized by ATP
The experimental conditions for the preparation of typical samples are described in the Experimental section and in Table 1. Our experiments indicate that ATP plays an important role in the stabilization of amorphous calcium carbonate. We investigated the stability of ACC in the presence of ATP and the effect of ATP concentration on the phase and morphology of calcium carbonate. The X-ray powder diffraction (XRD) pattern (Figure 1a) shows that the calcium carbonate control sample prepared in the absence of ATP at room temperature for 1 h consisted of a mixture of vaterite and calcite. However, in the presence of ATP even at a low concentration (1 mM), the product obtained for 1 h consisted of single-phase vaterite, and the XRD diffraction peaks of vaterite were widened (Figure 1b), this result may be explained by the hybridization of ATP molecules with ACC in the product. The ATP contents in the as-prepared samples are provided in Table 1. By increasing the ATP concentration to 2.5 mM and higher, the crystallization of amorphous calcium carbonate was completely inhibited, and the highly stable ACC was formed, indicating the important role of ATP in the stabilization of ACC in aqueous solution. Our experiments show that the yields of the products were in the range of 80–90%. Our previous work[35] shows that ATP molecules are stable and cannot hydrolyze to form phosphate ions in aqueous soluScheme 1. Schematic illustration of the formation of highly stable ACC nanospheres prepared tion at room temperature, and calcium phosphate cannot be obtained at room by using ATP as the stabilizer.
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© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
small 2014, 10, No. 10, 2047–2056
ATP-Stabilized Amorphous Calcium Carbonate Nanospheres and Their Application in Protein Adsorption
Figure 1. XRD patterns of the samples synthesized using aqueous solutions containing CaCl2, Na2CO3 and ATP at room temperature with different ATP concentrations for different times: (a) the control sample obtained without ATP, 1 h; (b) 1 mM ATP, 1 h; (c) 2.5 mM ATP, 1 h; (d) 4 mM ATP, 1 h; (e) 5 mM ATP, 1 h; (f) 5 mM ATP, 120 h.
temperature. Calcium phosphate is formed under the microwave-hydrothermal conditions at elevated temperatures (e.g. 120 °C). In this work, the syntheses of the products are carried out using aqueous solutions containing CaCl2, Na2CO3 and ATP at room temperature, therefore, no calcium phosphate forms in the product. For the synthesis of highly stable ATP-stabilized ACC nanospheres, when an aqueous solution of Na2CO3 is added into an aqueous solution of CaCl2 in the presence of ATP, CaCO3 is formed according to the following chemical reaction at room temperature: CaCl 2 + Na 2 CO 3 = CaCO 3 ↓ + 2NaCl
Figure 2. FTIR spectra of pure ATP and the samples synthesized using aqueous solutions containing CaCl2, Na2CO3 and ATP at room temperature with different ATP concentrations for different times: (a) the control sample obtained in the absence of ATP, 1 h; (b) 1 mM ATP, 1 h; (c) 2.5 mM ATP, 1 h; (d) 4 mM ATP, 1 h; (e) 5 mM ATP, 1 h; (f) 5 mM ATP, 120 h. small 2014, 10, No. 10, 2047–2056
In order to investigate the stability of the ATP-stabilized ACC, the aqueous solution containing the as-prepared ACC was stirred at room temperature for 120 h, and the product was still composed of ACC (Figure 1f), indicating that the as-prepared ACC has a high stability in aqueous solution for a relatively long period of time (at least 5 days). The FTIR spectra of the as-prepared samples are shown in Figure 2. Figure 2a shows that the calcium carbonate control sample prepared in the absence of ATP had a broad ν3 absorption band at around 1485 cm−1, a strong ν2 absorption band at 877 cm−1 (out-plane bending of CO32−) and a relatively weak ν4 absorption band at 746 cm−1 (in-plane bending of CO32−), which are characteristic of vaterite. However, the samples prepared in the presence of ATP showed a broad absorption band at about 3400 cm−1, which is assigned to the absorbed water, and the ν3 absorption band was split into two peaks at 1485 cm−1 and 1425 cm−1. For the ATP-stabilized ACC, the ν2 absorption peak shifted to 868 cm−1.[39,40] The samples prepared in the presence of ATP exhibited the PO43− absorption peaks located at around 1120 cm−1 and 560 cm−1,[41] together with the peaks at 1655 cm−1 and 1257 cm−1 originating from ATP molecules. The morphologies of the samples synthesized using aqueous solutions containing CaCl2, Na2CO3 and ATP at room temperature at different ATP concentrations were observed by scanning electron microscopy (SEM) (Figure 3) and transmission electron microscopy (TEM) (Figure 4). From Figure 4a, one can see that the calcium carbonate control sample prepared in the absence of ATP was composed of vaterite particles as the main product and polyhedral calcite crystals as a minor product, and the sizes of both components were in the micrometre scale. The selectedarea electron diffraction (SAED) pattern (Figure 4a) indicates high crystallinity of the control sample. In the presence of ATP even at a low concentration (1 mM), the polyhedral calcite crystals disappeared and the vaterite particles also became an ellipsoid shape (Figures 3b and 4b), the SAED pattern indicates poor crystallinity of vaterite particles. When the ATP concentration was further increased to 2.5 mM and higher, the vaterite particles completely disappeared, and the ACC nanospheres formed (Figures 3c–f and 4c–e), the SAED patterns (Figure 4c–e) also confirm that they were amorphous. It has been found that the size and morphology of the ACC nanospheres had no obvious change in aqueous solution even after vigorous stirring at room temperature for 120 h (Figures 3f and 4e). These results indicate that the ATP biomolecules have the ability to stabilize the ACC phase in aqueous solution for a long period of time. Figure 5 shows the UV-vis absorption spectra of the reaction system in the presence of ATP before the reaction and after the reaction for 1 min, exhibiting the characteristic absorption peak of ATP at about 258 nm, and the absorbance at 258 nm had a linear relationship with the ATP concentration in the range of 0–50 µg mL−1. The absorbance of ATP at 258 nm after 1 min reaction obviously decreased compared with that before the reaction (Figure 5), indicating that the formation of calcium carbonate was very fast. When the reaction time was further increased, the utilization percentage of ATP remained almost constant
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Figure 3. SEM micrographs of the samples synthesized using aqueous solutions containing CaCl2, Na2CO3 and ATP at different ATP concentrations at room temperature for different times: (a) the control sample obtained in the absence of ATP, 1 h; (b) 1 mM ATP, 1 h; (c) 2.5 mM ATP, 1 h; (d) 4 mM ATP, 1 h; (e) 5 mM ATP, 1 h; (f) 5 mM ATP, 120 h.
(Figure 6a). The utilization percentage is defined as: (Initial concentration – concentration after the reaction)/Initial concentration × 100%. The utilization percentages of phosphorus element (Figure 6b) originated from ATP were measured by inductively coupled plasma (ICP) analysis. The results of ICP analysis showed the same trend with those measured by UV-vis. It has been found that the utilization percentage of ATP decreased with increasing ATP concentration (Figure 6c). However, the utilization percentages of Ca element (Figure 6d) measured by ICP were similar regardless of the ATP concentration. The thermal stability of the calcium carbonate control sample prepared in the absence of ATP and the ATP-stabilized ACC nanospheres was measured by thermogravimetric (TG) analysis, and the results are shown in Figure 7. The calcium carbonate control sample had no obvious weight loss below 600 °C, indicating that the control sample had no
structural water, the decomposition temperatures of calcium carbonate were in the range of 600–850 °C, and the decomposition product after TG analysis was a single phase of calcium oxide. For the ATP-stabilized ACC nanospheres obtained using 5 mM ATP for 1 h at room temperature, the TG curve can be divided into four stages. The weight loss below 130 °C is assigned to the absorbed water, and the structural water loss was between 130–250 °C. The weight loss between 250–450 °C is attributed to the decomposition of ATP. When the temperature was higher than 450 °C, the weight loss is mainly assigned to the decomposition of calcium carbonate. Finally, the sample completely transformed to apatite at above 1000 °C. Figure 8 shows the N2 adsorption-desorption isotherms and pore size distribution curve of the ATP-stabilized ACC nanospheres prepared with 5 mM ATP at room temperature for 1 h. The as-prepared ATP-stabilized ACC nanospheres had a nanoporous structure with an average pore size of 32.51 nm. The Brunauer–Emmett–Teller (BET) specific surface area and the corresponding BJH desorption cumulative pore volume (Vp) of the ATP-stabilized ACC nanospheres were 70.92 m2 g−1 and 0.86 cm3 g−1, respectively. The relatively large SBET and Vp are favorable for applications in protein adsorption, which provide a large number of active sites and physical space for protein adsorption.
2.2. Degradation of ATP-Stabilized ACC Nanospheres in PBS Solution
Figure 4. TEM micrographs and SAED patterns (insets) of the samples synthesized using aqueous solutions containing CaCl2, Na2CO3 and ATP at room temperature at different ATP concentrations for different times: (a) the control sample obtained in the absence of ATP, 1 h; (b) 1 mM ATP, 1 h; (c) 2.5 mM ATP, 1 h; (d) 5 mM ATP, 1 h; (e) 5 mM ATP, 120 h.
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The degradation behaviors of ATP-stabilized ACC nanospheres and the calcium carbonate control sample in PBS solutions with different pH values were investigated, and the results are shown in Figure 9. The pH value had an obvious effect on the Ca2+ ion dissolution rate
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
small 2014, 10, No. 10, 2047–2056
ATP-Stabilized Amorphous Calcium Carbonate Nanospheres and Their Application in Protein Adsorption
Figure 5. UV-vis absorption spectra: the aqueous solution containing CaCl2 and ATP before the reaction; and aqueous solution containing CaCl2, Na2CO3 and ATP after the reaction for 1 min.
which represents the degradation rate of the sample, and both samples had a higher degradation rate at a lower pH value. For the calcium carbonate control sample obtained in the absence of ATP (Figure 9a), the percentage of the dissolved Ca2+ ions was relatively high at the beginning, and it gradually decreased and reached an equilibrium. As a whole, the percentage of the Ca2+ ions dissolved from the calcium carbonate control sample was very low (
ATP-Stabilized Amorphous Calcium Carbonate Nanospheres and Their Application in Protein Adsorption Chao Qi, Ying-Jie Zhu,* Bing-Qiang Lu, Xin-Yu Zhao, Jing Zhao, Feng Chen, and Jin Wu
Calcium carbonate is a common substance found in rocks worldwide, and is the main biomineral formed in shells of marine organisms and snails, pearls and eggshells. Amorphous calcium carbonate (ACC) is the least stable polymorph of calcium carbonate, which is so unstable under normal conditions that it is difficult to be prepared in vitro because it rapidly crystallizes to form one of the more stable polymorphs in aqueous solution. Herein, we report the successful synthesis of highly stable ACC nanospheres in vitro using adenosine 5′-triphosphate disodium salt (ATP) as a stabilizer. The effect of ATP on the stability of ACC nanospheres is investigated. Our experiments show that ATP plays an unique role in the stabilization of ACC nanospheres in aqueous solution. Moreover, the as-prepared ACC nanospheres are highly stable in phosphate buffered saline for a relatively long period of time (12 days) even under relatively high concentrations of calcium and phosphate ions. The cytotoxicity tests show that the as-prepared highly stable ACC nanospheres have excellent biocompatibility. The highly stable ACC nanospheres have high protein adsorption capacity, implying that they are promising for applications in biomedical fields such as drug delivery and protein adsorption.
1. Introduction Calcium carbonate (CaCO3), an ubiquitous and important biomineral in biological systems,[1,2] has aroused enormous interest because it is widely used as a model system for studying the biomimetic processes that are very common and important phenomena in nature.[3–6] Organisms have the ability of biomineralization to form a composite material composed of calcium carbonate embedded in the organic matrix,[7–9] which inspires researchers to synthesize calcium C. Qi, Prof. Dr. Y.-J. Zhu, Dr. B.-Q. Lu, X.-Y. Zhao, J. Zhao, F. Chen, Dr. J. Wu State Key Laboratory of High Performance Ceramics and Superfine Microstructure Shanghai Institute of Ceramics Chinese Academy of Sciences Shanghai 200050, P. R. China Tel: 0086-21-52412616; Fax: 0086-21-52413122 E-mail: [email protected] DOI: 10.1002/smll.201302984 small 2014, 10, No. 10, 2047–2056
carbonate in vitro. Calcium carbonate occurs naturally in three anhydrous crystalline polymorphs (calcite, aragonite and vaterite in the order of decreasing stability), two hydrated metastable forms (monohydrocalcite and calcium carbonate hexahydrate), and an unstable amorphous calcium carbonate (ACC) phase.[10–12] Among these polymorphs, ACC is the most important phase because it plays a pivotal role in the process of biomineralization for other phases and is the precursor to the crystalline polymorphs.[13,14] ACC has several potential biological functions due to its high solubility relative to the crystalline phases. On the other hand, the bioavailability of ACC, commonly used medicinally as a calcium supplement or as an antacid, is roughly 40% more bioavailable than the crystalline calcium carbonate with only around 20–30%.[15] Due to lower stability and higher solubility of ACC compared with the crystalline phases of calcium carbonate, it is difficult to produce pure ACC in vitro, and ACC will crystallize rapidly to form one of the more stable polymorphs in aqueous solution.[16] However, the highly stable ACC, stabilized by specific biological macromolecules which is a
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
wileyonlinelibrary.com
2047
full papers
C. Qi et al.
key principle in the nature of biomineralization,[17–20] has been found to exist in living organisms such as in ascidian skeleton, plant cystoliths, spicules from sponge and mollusk shells.[21,22] Inspired by biomineralization, many researchers have devoted to the design and synthesis of stabilized ACC with some additives such as biomolecules and organic molecules under mild conditions in vitro.[23–26] For example, Huang et al.[27] managed to stabilize ACC for several months using polyacrylic acid. In addition, the inorganic ions such as magnesium ions, phosphates as well as silica are also capable of stabilizing ACC for a certain period.[28–32] Freeze-drying can also produce stable ACC.[33] Bentov et al.[34] reported that phosphorylated amino acids could produce ACC with long-term stability. However, only a few methods have been reported for the synthesis of ACC which is stable in aqueous solution for more than several weeks. In order to understand the biomineralization mechanism of calcium carbonate, it is necessary to explore the effects of biomolecules on biomineralization. Adenosine 5′-triphosphate (ATP) is the most common energy carrier of the cell in the biological system, and plays an important role in various life activities for transporting chemical energy within cells for metabolism. Previous works indicate that ATP can be used as a stabilizer for amorphous calcium phosphate to inhibit the crystallization process in aqueous solution by poisoning heterogeneous nucleation sites and binding to embryonic crystal nucleus.[35–37] Moreover, ATP can strongly inhibit the formation of calcium carbonate crystals and induce deposition of amorphous calcium carbonate.[38] Herein, we report the successful synthesis of highly stable ACC nanospheres in vitro using ATP as the stabilizer (Scheme 1). The effect of ATP biomolecules on the stability of ACC nanospheres in aqueous solution is investigated. Moreover, the as-prepared ACC nanospheres are highly stable in phosphate buffered saline (PBS) for a relatively long period of time (12 days) even at relatively high concentrations of calcium ions and phosphate ions, which is important for the investigation of the biomineralization processes. Furthermore, the
Table 1. Experimental conditions for the preparation of typical samples using aqueous solutions containing CaCl2, Na2CO3 and ATP (pH 9) and the ATP contents of the products. Sample No.
ATP concentration [mM]
Stirring time at room temp. [h]
ATP content [wt.%]
1
–
1
–
2
1
1
18.373
3
2.5
1
26.738
4
4
1
37.561
5
5
1
49.565
6
5
120
43.589
cytotoxicity tests show that the as-prepared highly stable ACC nanospheres have excellent biocompatibility. The highly stable ACC nanospheres can be used as the excellent adsorbent with high protein adsorption capacity, indicating that they are promising for applications in biomedical fields such as drug delivery and protein adsorption.
2. Results and Discussion 2.1. Formation and Characterization of ACC Nanospheres Stabilized by ATP
The experimental conditions for the preparation of typical samples are described in the Experimental section and in Table 1. Our experiments indicate that ATP plays an important role in the stabilization of amorphous calcium carbonate. We investigated the stability of ACC in the presence of ATP and the effect of ATP concentration on the phase and morphology of calcium carbonate. The X-ray powder diffraction (XRD) pattern (Figure 1a) shows that the calcium carbonate control sample prepared in the absence of ATP at room temperature for 1 h consisted of a mixture of vaterite and calcite. However, in the presence of ATP even at a low concentration (1 mM), the product obtained for 1 h consisted of single-phase vaterite, and the XRD diffraction peaks of vaterite were widened (Figure 1b), this result may be explained by the hybridization of ATP molecules with ACC in the product. The ATP contents in the as-prepared samples are provided in Table 1. By increasing the ATP concentration to 2.5 mM and higher, the crystallization of amorphous calcium carbonate was completely inhibited, and the highly stable ACC was formed, indicating the important role of ATP in the stabilization of ACC in aqueous solution. Our experiments show that the yields of the products were in the range of 80–90%. Our previous work[35] shows that ATP molecules are stable and cannot hydrolyze to form phosphate ions in aqueous soluScheme 1. Schematic illustration of the formation of highly stable ACC nanospheres prepared tion at room temperature, and calcium phosphate cannot be obtained at room by using ATP as the stabilizer.
2048 www.small-journal.com
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
small 2014, 10, No. 10, 2047–2056
ATP-Stabilized Amorphous Calcium Carbonate Nanospheres and Their Application in Protein Adsorption
Figure 1. XRD patterns of the samples synthesized using aqueous solutions containing CaCl2, Na2CO3 and ATP at room temperature with different ATP concentrations for different times: (a) the control sample obtained without ATP, 1 h; (b) 1 mM ATP, 1 h; (c) 2.5 mM ATP, 1 h; (d) 4 mM ATP, 1 h; (e) 5 mM ATP, 1 h; (f) 5 mM ATP, 120 h.
temperature. Calcium phosphate is formed under the microwave-hydrothermal conditions at elevated temperatures (e.g. 120 °C). In this work, the syntheses of the products are carried out using aqueous solutions containing CaCl2, Na2CO3 and ATP at room temperature, therefore, no calcium phosphate forms in the product. For the synthesis of highly stable ATP-stabilized ACC nanospheres, when an aqueous solution of Na2CO3 is added into an aqueous solution of CaCl2 in the presence of ATP, CaCO3 is formed according to the following chemical reaction at room temperature: CaCl 2 + Na 2 CO 3 = CaCO 3 ↓ + 2NaCl
Figure 2. FTIR spectra of pure ATP and the samples synthesized using aqueous solutions containing CaCl2, Na2CO3 and ATP at room temperature with different ATP concentrations for different times: (a) the control sample obtained in the absence of ATP, 1 h; (b) 1 mM ATP, 1 h; (c) 2.5 mM ATP, 1 h; (d) 4 mM ATP, 1 h; (e) 5 mM ATP, 1 h; (f) 5 mM ATP, 120 h. small 2014, 10, No. 10, 2047–2056
In order to investigate the stability of the ATP-stabilized ACC, the aqueous solution containing the as-prepared ACC was stirred at room temperature for 120 h, and the product was still composed of ACC (Figure 1f), indicating that the as-prepared ACC has a high stability in aqueous solution for a relatively long period of time (at least 5 days). The FTIR spectra of the as-prepared samples are shown in Figure 2. Figure 2a shows that the calcium carbonate control sample prepared in the absence of ATP had a broad ν3 absorption band at around 1485 cm−1, a strong ν2 absorption band at 877 cm−1 (out-plane bending of CO32−) and a relatively weak ν4 absorption band at 746 cm−1 (in-plane bending of CO32−), which are characteristic of vaterite. However, the samples prepared in the presence of ATP showed a broad absorption band at about 3400 cm−1, which is assigned to the absorbed water, and the ν3 absorption band was split into two peaks at 1485 cm−1 and 1425 cm−1. For the ATP-stabilized ACC, the ν2 absorption peak shifted to 868 cm−1.[39,40] The samples prepared in the presence of ATP exhibited the PO43− absorption peaks located at around 1120 cm−1 and 560 cm−1,[41] together with the peaks at 1655 cm−1 and 1257 cm−1 originating from ATP molecules. The morphologies of the samples synthesized using aqueous solutions containing CaCl2, Na2CO3 and ATP at room temperature at different ATP concentrations were observed by scanning electron microscopy (SEM) (Figure 3) and transmission electron microscopy (TEM) (Figure 4). From Figure 4a, one can see that the calcium carbonate control sample prepared in the absence of ATP was composed of vaterite particles as the main product and polyhedral calcite crystals as a minor product, and the sizes of both components were in the micrometre scale. The selectedarea electron diffraction (SAED) pattern (Figure 4a) indicates high crystallinity of the control sample. In the presence of ATP even at a low concentration (1 mM), the polyhedral calcite crystals disappeared and the vaterite particles also became an ellipsoid shape (Figures 3b and 4b), the SAED pattern indicates poor crystallinity of vaterite particles. When the ATP concentration was further increased to 2.5 mM and higher, the vaterite particles completely disappeared, and the ACC nanospheres formed (Figures 3c–f and 4c–e), the SAED patterns (Figure 4c–e) also confirm that they were amorphous. It has been found that the size and morphology of the ACC nanospheres had no obvious change in aqueous solution even after vigorous stirring at room temperature for 120 h (Figures 3f and 4e). These results indicate that the ATP biomolecules have the ability to stabilize the ACC phase in aqueous solution for a long period of time. Figure 5 shows the UV-vis absorption spectra of the reaction system in the presence of ATP before the reaction and after the reaction for 1 min, exhibiting the characteristic absorption peak of ATP at about 258 nm, and the absorbance at 258 nm had a linear relationship with the ATP concentration in the range of 0–50 µg mL−1. The absorbance of ATP at 258 nm after 1 min reaction obviously decreased compared with that before the reaction (Figure 5), indicating that the formation of calcium carbonate was very fast. When the reaction time was further increased, the utilization percentage of ATP remained almost constant
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Figure 3. SEM micrographs of the samples synthesized using aqueous solutions containing CaCl2, Na2CO3 and ATP at different ATP concentrations at room temperature for different times: (a) the control sample obtained in the absence of ATP, 1 h; (b) 1 mM ATP, 1 h; (c) 2.5 mM ATP, 1 h; (d) 4 mM ATP, 1 h; (e) 5 mM ATP, 1 h; (f) 5 mM ATP, 120 h.
(Figure 6a). The utilization percentage is defined as: (Initial concentration – concentration after the reaction)/Initial concentration × 100%. The utilization percentages of phosphorus element (Figure 6b) originated from ATP were measured by inductively coupled plasma (ICP) analysis. The results of ICP analysis showed the same trend with those measured by UV-vis. It has been found that the utilization percentage of ATP decreased with increasing ATP concentration (Figure 6c). However, the utilization percentages of Ca element (Figure 6d) measured by ICP were similar regardless of the ATP concentration. The thermal stability of the calcium carbonate control sample prepared in the absence of ATP and the ATP-stabilized ACC nanospheres was measured by thermogravimetric (TG) analysis, and the results are shown in Figure 7. The calcium carbonate control sample had no obvious weight loss below 600 °C, indicating that the control sample had no
structural water, the decomposition temperatures of calcium carbonate were in the range of 600–850 °C, and the decomposition product after TG analysis was a single phase of calcium oxide. For the ATP-stabilized ACC nanospheres obtained using 5 mM ATP for 1 h at room temperature, the TG curve can be divided into four stages. The weight loss below 130 °C is assigned to the absorbed water, and the structural water loss was between 130–250 °C. The weight loss between 250–450 °C is attributed to the decomposition of ATP. When the temperature was higher than 450 °C, the weight loss is mainly assigned to the decomposition of calcium carbonate. Finally, the sample completely transformed to apatite at above 1000 °C. Figure 8 shows the N2 adsorption-desorption isotherms and pore size distribution curve of the ATP-stabilized ACC nanospheres prepared with 5 mM ATP at room temperature for 1 h. The as-prepared ATP-stabilized ACC nanospheres had a nanoporous structure with an average pore size of 32.51 nm. The Brunauer–Emmett–Teller (BET) specific surface area and the corresponding BJH desorption cumulative pore volume (Vp) of the ATP-stabilized ACC nanospheres were 70.92 m2 g−1 and 0.86 cm3 g−1, respectively. The relatively large SBET and Vp are favorable for applications in protein adsorption, which provide a large number of active sites and physical space for protein adsorption.
2.2. Degradation of ATP-Stabilized ACC Nanospheres in PBS Solution
Figure 4. TEM micrographs and SAED patterns (insets) of the samples synthesized using aqueous solutions containing CaCl2, Na2CO3 and ATP at room temperature at different ATP concentrations for different times: (a) the control sample obtained in the absence of ATP, 1 h; (b) 1 mM ATP, 1 h; (c) 2.5 mM ATP, 1 h; (d) 5 mM ATP, 1 h; (e) 5 mM ATP, 120 h.
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The degradation behaviors of ATP-stabilized ACC nanospheres and the calcium carbonate control sample in PBS solutions with different pH values were investigated, and the results are shown in Figure 9. The pH value had an obvious effect on the Ca2+ ion dissolution rate
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
small 2014, 10, No. 10, 2047–2056
ATP-Stabilized Amorphous Calcium Carbonate Nanospheres and Their Application in Protein Adsorption
Figure 5. UV-vis absorption spectra: the aqueous solution containing CaCl2 and ATP before the reaction; and aqueous solution containing CaCl2, Na2CO3 and ATP after the reaction for 1 min.
which represents the degradation rate of the sample, and both samples had a higher degradation rate at a lower pH value. For the calcium carbonate control sample obtained in the absence of ATP (Figure 9a), the percentage of the dissolved Ca2+ ions was relatively high at the beginning, and it gradually decreased and reached an equilibrium. As a whole, the percentage of the Ca2+ ions dissolved from the calcium carbonate control sample was very low (
