Carbonic Anhydrase-Assisted CaCO3 Film Deposition at the Air

Jan 7, 2011 - from the air. In the presence of carbonic anhy- drase, CaCO3 was deposited at the airrsolution interface by a reaction between calcium i...
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Carbonic Anhydrase-Assisted CaCO3 Film Deposition at the Air-Solution Interface Shichoon Lee, Seung Goo Lee, Donghoon Kwak, Jong-Hwan Park, and Kilwon Cho* Department of Chemical Engineering, Pohang University of Science and Technology, Pohang, 790-784, Korea

bS Supporting Information ABSTRACT: We observed that carbonic anhydrase lowered the surface energy of the aqueous solution by way of enhanced CO2 hydration from the air. In the presence of carbonic anhydrase, CaCO3 was deposited at the air-solution interface by a reaction between calcium ions in the solution and carbonate ions formed by the dissolution of CO2 from the air. This observation suggests that a carbonic anhydrase would facilitate CaCO3 crystal nucleation and growth at the interface via heterogeneous nucleation at the air-solution interface by controlling the surface energy of solution.

’ INTRODUCTION Many organisms deposit calcium carbonate or calcium phosphate minerals to form structural or protective materials. Biomineralization is a complicated biological process, but on which the principles involved in ceramic formation are applicable. Both levels of local supersaturation and a net interfacial energy are also key factors to control crystal nucleation and growth in ceramic formation.1 Control over these two factors permits evolution of specific morphology with desirable properties including thin film morphology on a substrate via heterogeneous nucleation. These films differ from those formed by precipitation due to homogeneous nucleation. Biomineralization favors heterogeneous nucleation and growth and forms specific morphology. A carbonic anhydrase (CA) is a ubiquitous enzyme and catalyzes CO2 hydration and dehydration of bicarbonates.2 Several studies have reported the involvement of CA in biomineralization as a protein for skeletogenesis in coralline sponges3 or as constitutive macromolecules.4 The presence of phosphorylated proteins,5 acidic proteins6 with carboxylic acids, and polysaccharides with sulfate groups are crucial to the process of biomineralization. Because these all convey an anionic character under the pH in biomineralization, these anionic groups are readily bound to soluble metallic cations, which control nucleation of calcium phosphate or calcium carbonate crystals. Rudloff showed that the change in degree of phosphorylation in the poly(ethylene oxide-methacrylate) block copolymer altered the surface tension of solutions and permitted control over the CaCO3 morphology.7 The surface energy at a substrate may vary as a function of the degree of protein phosphorylation to favor formation of the desirable biomineralizaed structures. These studies indicate that organisms may utilize the strategy associated with the surface r 2011 American Chemical Society

tension by control of the degree of anionic groups in the macromolecular chain or amount of anionic groups as well as the binding affinities of the anionic moieties toward metallic cations.8 However, few studies on the interfacial tension in biomineralization have been carried out. As a simple model study, a calcium chloride solution containing an acidic polymer, a basic component, and CA was introduced. Deposition of CaCO3 was induced by the reaction between the calcium ions in solution and the carbonate ions that had been generated by the decomposition of bicarbonate ions derived from hydrated CO2 from the air. The changes in surface tension were measured upon addition of CA and observed its effect on CaCO3 morphology by combining CA with anionic components in the presence of a basic buffered CaCl2 solution and CO2 sequestered from the ambient air.

’ EXPERIMENTAL SECTION A 10 mM CaCl2 solution containing 10 μg/mL bovine carbonic anhydrase II (CA, Sigma Aldrich) and basic and acidic components was prepared. Twenty milliliters of the solution was poured into a 5 cm Petri dish and stirred at around 250 rpm at room temperature, and the dish was covered by a plastic lid with several 2 mm diameter holes. The basic buffer component used was polyethylenimine (PEI) with molecular weights of 400. Poly(acrylic acid) (PAA; molecular weight 2000 g/mol) was used as an acidic polymer. The surface tension of the solution was measured with a Du Nouy’s tensiometer (Itoh 514) with pausing stirring before the Received: September 26, 2010 Revised: December 11, 2010 Published: January 7, 2011 2026

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The Journal of Physical Chemistry C CaCO3 layer appeared at the interface. The CaCO3 deposition layer appeared at the air-solution interface after about 4 h stirring by the reaction between calcium ions and carbonate ions from the CO2 dissolved directly from the atmosphere at room temperature. The deposited CaCO3 was collected on silicon wafers cleaned with piranha solution. [Caution: piranha solution is aggressive and explosive. Never mix piranha waste with solvents. Check the safety precautions before using it.] Field emission scanning electron microscopy (SEM, Hitachi S-4800) was used to investigate morphologies of the deposited CaCO3. The CaCO3 polymorphs were identified by reflection mode X-ray diffraction θ/2θ scans at the Pohang Accelerator Laboratory, Korea.

’ RESULTS AND DISCUSSION The addition of CA to the CaCl2 solution induced deposition of CaCO3 at the air-solution interface of a CaCl2 solution containing PEI and PAA under atmospheric conditions. Only in the presence of PEI in the CaCl2 solution was CaCO3 deposition not noticeable at the air-solution interface, but CaCO3 precipitation is noticeable on the bottom. The addition of CA in the presence of PEI caused CaCO3 deposition at the interface, and the formed CaCO3 was mixed with calcite and aragonite.9 The presence of CA and PAA in the CaCl2 solution produced a calcite layer at the air-solution interface (Figure 1). An increase in the concentration of PAA caused by the deposition was slow but generated a thicker film-like layer at the air-solution interface. At a 100 μg/mL PAA concentration, almost no precipitate was

Figure 1. CaCO3 deposited at the air-solution interface in Petri dishes by a reaction between calcium ions and carbonate ions derived from dissolved CO2. Experiments were performed with magnetic stirring in the presence of 10 μg/mL CA and 10 mg/mL PEI in 10 mM CaCl2 with several concentrations of PAA. (a) 10 μg/mL PAA: upper, 10 h; lower, 24 h. (b) 100 μg/mL PAA: upper, 10 h; lower, 24 h. (c) 100 μg/mL PAA: upper, 48 h in the absence of CA; lower, 24 h after the addition of CA (72 h aging time in total).

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observed at the bottom of the dish, but a thick opaque layer formed at the air-solution interface after 10 or 24 h aging time (Figure 1b). This observation indicated that in the presence of CA the increase in PAA concentration in the solution enhanced CaCO3 deposition at the air-solution interface but decreased precipitation on the bottom. The influence of CA on the CaCO3 depositions was investigated using solutions containing PEI and PAA either with or without CA. No noticeable deposition layer was observed at the air-solution interface except precipitation on the bottom even after 48 h in the absence of CA (upper, Figure 1c, stirred under atmospheric condition for 24 h and incubated at room temperature without stirring for 24 h). When CA and CaCl2 were supplemented into the solution and then the solution was stirred for 10 h, a film-like layer appeared in solution (lower, Figure 1c). These experiments suggested that CA facilitated CaCO3 deposition at the air-solution interface. Figure 2 shows a drop in the surface tensions of solutions containing CA. Even a water solution containing CA showed a drop in surface tension in 30 min after preparation (Figure 2b). No noticeable drop in surface tension occurred in solutions that did not contain CA (Figure 2a,c,d). The addition of CA to a CaCl2 solution containing PEI and PAA showed a drop in surface tension (Figure 2e,f). However, no drop in surface tension was observed in the CaCl2 solution containing CA if the solution was incubated without stirring at room temperature in a closed Petri dish (Supporting Information, Figure 1). Schaefer10,11observed the decrease in surface tension of epithelial cells by exposure of

Figure 2. Surface tension of solutions measured using a Du Nouy’s tensiometer under ambient conditions with or without 10 μg/mL CA: (a) purified water; (b) CA in purified water; (c) 10 mg/mL PEI; (d) 10 mg/mL PEI and 100 μg/mL PAA; (e) 30 mg/mL PEI, 10 μg/mL PAA with CA; (f) 10 mg/mL PEI, 100 μg/mL PAA with CA, in 10 mM CaCl2.

Scheme 1. Proposed Mechanism of CaCO3 Deposition at the Air-Solution Interfacea

a Illustration shows the accumulation of CO2, bicarbonates, and carbonate ions and deposition of CaCO3 layer at the interface by CO2 hydration in the presence of CA.

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dx.doi.org/10.1021/jp1091834 |J. Phys. Chem. C 2011, 115, 2026–2029

The Journal of Physical Chemistry C CO2, which caused morphological change of cells. CO2 is widely used in the separation and recovery process as a kind of solvent in the fields of chemical engineering and industries.12 These applications of CO2 are mainly attributed to its lowest surface tension as a solvent. It is no wonder that the increase in CO2 concentration at the interface by CA lowers the surface tension of the solution. Scheme 1 shows that zinc-containing CA enzyme hydrates CO2 to bicarbonates, and those further convert into carbonate ions in the presence of basic buffer. Hydration of CO2 accompanied acidification of the solution, preventing further hydration of CO2 (Supporting Information, Figure 2). But the solutions in this experiment contained sufficient quantities of basic PEI to buffer the protons generated during hydration, which can afford to produce a higher concentration of carbonate ions. The hydration process catalyzed by CA is buffermediated,12 and its activity depends on the nature of the buffer

Figure 3. Morphology of CaCO3 precipitated on the bottom in the absence of CA and in the presence of 10 mg/mL PEI and 100 μg/mL PAA in 10 mM CaCl2.

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used.12 CO2 hydration by CA and the generation of carbonates in a basic buffer primarily may occur at the air-solution interface, which enhances concentrations of these components at the interface. Enhanced concentrations of CO2, bicarbonates, and carbonate ions would reduce the surface tension at the air-solution interface. In addition, the CO2 hydration would enhance the local supersaturation of CaCO3. The degree of supersaturation and interfacial energy are dominant factors for crystal nucleation and growth. Bunker1 reported that a decrease in the interfacial surface tension influenced crystal nucleation to a much larger degree than changes in the level of supersaturation. The proper combination of reduced surface energy and degree of supersaturation induce heterogeneous nucleation, resulting in film deposition on the substrate, even at relatively low supersaturation levels that may not have produced homogeneous nucleation. The role of the cationic polymer PEI on CaCO3 formation was investigated. In the absence of PEI noticeable CaCO3 formed neither at the air-solution interface nor at the bottom. In the absence of PAA and CA, the presence of PEI gave a precipitation on the bottom. The supplement of CA in the solution containing PEI brought the deposition prevalent at the air-solution interface.9 Similar morphological developments were observed when ammonium hydroxide was used as a basic buffer instead of PEI. This suggests that basic components such as PEI and ammonium hydroxide play a basic buffer role in these experiments, leading to higher solution pH, which would influence the kinetics of CaCO3 formation. The morphology of the precipitate on the bottom was totally different from those of the deposits at the interface. Neither film nor orientated morphology was observed in the precipitate, but spheres commonly appeared on the bottom. In the absence of CA, CaCO3 precipitation was prevalent in the form of spheres on the bottom even in the presence of PAA, as shown in Figure 3. This indicates that the sphere morphology

Figure 4. Morphological developments of CaCO3 deposited at the air-solution interface at different concentrations of PEI in the presence of 10 μg/mL CA in 10 mM CaCl2 with 10 h. Surface and side views collected for varying amounts of PEI in the presence of 10 μg/mL PAA: (a) 30 mg/mL; (b) 20 mg/mL; (c) 10 mg/mL; (d) 5 mg/mL. 2028

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The Journal of Physical Chemistry C would be attributable to the homogeneous nucleation and growth in the solution. The addition of PAA into the solution containing CA and PEI gave calcite deposition at the air-solution interface. The soluble proteins involved in biomineralization are usually acidic and are known to control crystal nucleation and growth as well as to act as templates for the production of specific morphologies. The presence of a water-soluble acidic macromolecules or a change in its concentration alters the crystal morphology and orientation in the biomimetic CaCO3 synthesis.6,7,13,14 We used a soluble acidic polymer PAA, which is commonly used as a macromolecule bearing properties of an acidic protein in biomineralization, to develop controlled morphologies using this CA-assisted system. Figure 4 shows the morphological changes of the CaCO3 collected at the air-solution interface at varying concentrations of PEI in the presence of CA and PAA. At higher concentrations of PEI (30 mg/mL), large-area film deposited was transparent, sleek, and uniform (Figure 4a). The film was characterized as an amorphous calcium carbonate (ACC) by XRD studies (Supporting Information, Figure 3). The spherulites grew horizontally and impinged upon one another. The film thickness is around 10-20 nm (inset in Figure 4a). The preparation of nanosized high quality films with controlled morphology is challenging and has potentials in the fields of microelectronics as well as the conventional coating industry. Lowering the PEI concentration below 10 mg/mL produced a film surface that either was coarse or contained discrete splaying cones at the interface that was composed of nanofibers (Figure 4b-d and each inset). A similar morphological change was also observed at increasing concentration of the added PAA in the presence of the fixed amount of PEI and CA.9 The presence of CA enhanced the CaCO3 crystal nucleation and growth in the heterogeneous manner at the air-solution interface. These observations implied that CA may function as a surface-active agent at the interface, thereby providing a functional interface for mineral deposition. This system would be a good model for the biomineralization process, which involves heterogeneous crystal nucleation and growth.

’ CONCLUSIONS We found that CA decreased the solution surface energy by enhancing hydration of ambient atmospheric CO2. The decrease in surface energy facilitated heterogeneous crystal nucleation and growth at the air-solution interface. CaCO3 was deposited at the air-solution interface in the presence of CA via the reaction of calcium ions with carbonate ions derived from dissolved CO2. This suggests that the CA-assisted system induces heterogeneous nucleation and growth of calcium carbonate at the air-solution interface. CaCO3 with various morphologies, such as a film with nanometer thickness, nanofibers, or cone-shaped aggregates, developed with the addition of soluble acidic polymers to the solutions. The CA-assisted system devised may be used to develop a variety of nanoarchitectures, and the system provides a tool for studying biomineralization from carbonate ions formed by the dissolution of CO2 from the air.

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’ AUTHOR INFORMATION Corresponding Author

*Tel: 82-54-279-2270. Fax: 82-54-279-8298. E-mail: [email protected].

’ ACKNOWLEDGMENT This work was supported by a grant from the Center for Nanostructural Materials Technology (2010K000284) under the 21st Century Frontier R&D Programs, and Green Science Program of the Research Institute of Industrial Science and Technology. ’ REFERENCES (1) Bunker, B. C.; Rieke, P. C.; Tarasevich, B. J.; Campbell, A. A.; Fryxell, G. E.; Graff, G. L.; Song, L.; Liu, J.; Virden, J. W.; Mcvay, G. L. Science 1994, 264, 48–55. (2) Henry, R. P. Annu. Rev. Physiol. 1996, 58, 523–538. (3) Jackson, D. J.; Macis, L.; Reitner, J.; Degnan, B. M.; W€orheide, G. Science 2007, 316, 1893–1895. (4) Miyamoto, H.; Miyashita, T.; Okushima, M.; Nakano, S.; Morita, T.; Matsushiro, A. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 9657–9660. (5) He, G.; Ramachandran, A.; Dahl, T.; George, S.; Schultz, D.; Cookson, D.; Veis, A.; George, A. J. Biol. Chem. 2005, 280, 33109– 33114. (6) Boskey, A. L.; Maresca, M.; Doty, S.; Sabsay, B.; Veis, A. Bone Mineral. 1990, 11, 55–65. (7) Rudloff, J.; C€olfen, H. Langmuir 2004, 20, 991–996. (8) (a) Han, J. T.; Xu, X.; Kim, D. H.; Cho, K. Adv. Funct. Mater. 2005, 15, 475–480. (b) Xu, A. W.; Dong, W. F.; Antonietti, M.; C€olfen, H. Adv. Mater. 2005, 17, 2217–2221. (c) Yu, S. H.; C€olfen, H.; Hartman, J.; Antonietti, M. Adv. Funct. Mater. 2002, 12, 541–545. (9) Lee, S.; Park, J.-H.; Kwak, D.; Cho, K. Cryst. Growth Des. 2010, 10, 851–855. (10) Schaefer, K. E.; Avery, M. E.; Bensch, K. J. Clin. Invest. 1964, 43, 2080–2093. (11) Jianxin, P.; Yiyang, L. Phys. Chem. Liq. 2009, 47, 267–273. (12) (a) Khalifah, R. G. Proc. Natl. Acad. Sci. U.S.A. 1973, 70, 1986– 1989. (b) Khalifah, R. G. J. Biol. Chem. 1971, 246, 2561–2573. (13) Heywood, B. R.; Mann, S. Langmuir 1992, 8, 1492–1498. (14) (a) Mei, L.; Mann, S. Adv. Funct. Mater. 2002, 12, 773–779. (b) Mann, S.; C€olfen, H. Angew. Chem., Int. Ed. 2003, 42, 2350–2365.

’ ASSOCIATED CONTENT

bS

Supporting Information. Figures of surface tension measured and X-ray diffraction data. This material is available free of charge via the Internet at http://pubs.acs.org. 2029

dx.doi.org/10.1021/jp1091834 |J. Phys. Chem. C 2011, 115, 2026–2029