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Formation of Calcium Carbonate Crystal Using Phospholipid Monolayer Template Under Ambient Condition Prabir Pal,† Tapanendu Kamilya,§,† Somobrata Acharya,‡ and G. B. Talapatra*,† Department of Spectroscopy, Indian Association for the CultiVation of Science, JadaVpur, Kolkata-700 032, India, and Center for AdVanced Materials, Indian Association for the CultiVation of Science, JadaVpur, Kolkata-700 032, India ReceiVed: March 25, 2010; ReVised Manuscript ReceiVed: April 9, 2010
Formation and assembly of monodispersed calcite (CaCO3) dots of diameters 6-10 nm using a zwitterionic phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) monolayer as template at a two-dimensional interface is reported here. We utilized penetration of Ca2+ at lower surface pressure (π) into the DPPC monolayer, governed by a single exponential association method. In situ surface pressure (π)-area (A) isotherms as well as compressibility (β) analysis reveal a positive association of Ca2+ with DPPC. The natural exposure of the resultant monolayer to air results in formation of single crystalline CaCO3 nanodots. HRTEM and SAED techniques have been used to study the crystal structure of the resultant CaCO3 nanodots incorporated within the lipid monolayer.
Biological systems or organisms are capable of producing and controlling the formation of biominerals that are superior to their geological counterparts in many aspects. The process of biomineralization provides a structural framework and regulates dynamic processes such as nucleation site, direction of orientation, growth, and assembly of crystals,1-3 etc. In the process of biomineralization, biomolecules form templates to synthesize new materials. The underlying mechanisms associated with biomineralization are molecular recognition at the organic-inorganic interface associated with electrostatic interactions and stereochemical and geometrical matching.1-6 The process of biomineralization has inspired many researchers to investigate template-directed crystal formation at condensed surfactant thin films. Recent reviews7-9 about biomineralization, including nanomaterials formation and fabrication at interfacial media, are worth mentioning here. The syntheses of calcium carbonate (CaCO3) nanocrystals are of considerable current interest owing to their potential biomedical applications in drug delivery material10 and gene therapy for cancer.11 Among the three anhydrous crystalline phases (calcite, aragonite, and vaterite) of natural CaCO3, calcite is thermodynamically the most stable, whereas vaterite is the most unstable. Well-ordered structured calcite and aragonite are very common in biologically produced minerals. Calcite occurs as a biomineral, and it happens to be the major constituent of pancreatic calculi12-14 and a constituent of gall bladder stones.15 To mimic and to recognize the biological mineralization of CaCO3, a number of biomimitic templates, such as a Langmuir monolayer, self-assembled monolayer, lipid bilayer stacks, dynamic liquid-liquid interfaces, vesicles and functionalized micropatterned surfaces for its synthesis, have been adopted.16-21 Among these templates, the Langmuir monolayer has a great
advantage in illustrating the nature of this biomineralization process as well as in situ observation of the interaction of inorganic materials with biomolecules.22 In the Langmuir monolayer, the well-ordered and highly oriented two-dimensional fabrications of lipid molecules at the air/water interface23,24 acts as an influential template for wellcontrolled nucleation and growth of biominerals providing the microspace.19 In addition, the mobility of the molecules has an immense influence on (1) the homogeneity and nucleation density of the crystallization and (2) the morphology of the overgrowth.25 However, a major challenge remaining is to synthesize CaCO3 nanocrystals with dimensions less than 20 nm by using a phospholipid monolayer at ambient conditions. In the present case, organic template films of zwitterionic phospholipids (1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)) were fabricated using a Langmuir trough. We investigated crystallization of nanosized single calcite crystals on a single-layered LB film template of DPPC, transferred from the normal-air/calcium chloride (CaCl2)-containing water interface (without using excess CO2 or any carbonate salt). We varied the composition and physical state of the film by adjusting the compression speed, lifting speed, lifting surface pressure, and CaCl2 concentrations; hence, changing the interaction with the subphase solution. The surface pressure-area isotherms as well as compressibility analysis show association of Ca2+ is feasible at a lower surface pressure of the DPPC monolayer. Followed by the association of Ca2+ with DPPC, the exposure to natural atmosphere is responsible for the formation of well-defined CaCO3 crystals having 6-10 nm diameters. Natural formations of such nanocrystals have useful implications in understanding biomineralization processes and in fabricating nanocomposite materials.
* Corresponding author. Phone: +91-33-24734971. Fax: +91-3324732805. E-mail:
[email protected]. † Department of Spectroscopy. ‡ Center for Advanced Materials. § Present address: Department of Physics, Narajole Raj College, Narajole, Paschim Medinipur -721 211, India.
2. Experimental Section 2.1. Materials. 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine and calcium chloride were purchased from Sigma and Merck Chemical Co., respectively. Those chemicals were used as
1. Introduction
10.1021/jp102696s 2010 American Chemical Society Published on Web 04/19/2010
Formation of Calcium Carbonate Crystal received without further purification. Spectral grade chloroform (SRL, India) was used to prepare a 1 mM solution of DPPC. 2.2. Incorporation of Ca2+ into the DPPC Monolayer. To study the adsorption kinetics of the Ca2+ into the DPPC monolayer, the pure DPPC monolayer was first precompressed to the requisite pressure, and then the aqueous solution of CaCl2 was injected into the subphase to attain the required final concentration. 2.3. DPPC Monolayer Preparation. Chloroform solution of DPPC was spread on a triple-distilled water subphase of a Teflon-bar-barrier type LB trough (model 2007DC, Apex Instruments Co., India), deionized with a Milli-Q water purification system from Millipore (Billerica, MA). The pH and resistivity of the distilled water were 6.8 and 18.2 MΩ cm, respectively. After a delay of ∼20 min to allow the solvent to evaporate, the monolayer was slowly compressed to the required pressures with a compression speed of 1 Å2/(molecule min). The surface pressure was measured using a Wilhelmy balance. All experiments were performed at a temperature of 26 ( 1 °C unless otherwise mentioned. 2.4. Preparation of Ca2+ Mixed DPPC Monolayer. For the preparation of Ca2+-DPPC LB monolayer, we mixed the CaCl2 with a water subphase. DPPC solution was spread in the surface, and barriers were compressed to obtain a monolayer at a particular pressure. For the Ca2+ penetration kinetics study, CaCl2 stock solution was injected beneath the precompressed DPPC monolayer on the pure water subphase. 2.5. Monolayer Transfer. The monolayer was then transferred carefully with a speed of 5 mm/min onto hydrophilic glass coverslips or silicon wafers, which were immersed in the subphase before monolayer spreading. The details of the procedure of cleaning of the quartz slides and glass coverslips are described elsewhere.23,26 2.6. Study of Surface Morphology. High-resolution field emission scanning electron microscope (FE-SEM, model JEOL JSM-6700 F) with use range: 0.5-30 kV with a lateral resolution in the range of 2.2-1.2 nm was employed to measure the surface morphology of all transferred films. Energy-dispersive X-ray (EDX) analysis was also performed using this instrument. 2.7. HR-TEM Analysis. The Ca2+-DPPC mixed film was transferred onto a carbon-coated, 3 mm diameter, copper electron microscope grid for transmission electron microscopy (TEM, JEOL). Selected area electron diffraction (SAID) was also performed using this instrument. 2.8. Simulation Study. A simulation study for crystal structure was performed by the software Crystal Maker, Version 8 using the bulk parameters derived from the JCPDS 86-0174 data bank. 2. Results and Discussion 3.1. π-A Isotherm. Surface pressure (π)-area (A) compression isotherms of a Langmuir monolayer of DPPC in pure water subphase and in a water subphase containing CaCl2 with different concentrations (5 and 25 mM) shows a plateaulike region (Figure 1). This plateau originates from phase coexistence, associating with the first-order phase transition between the liquid-expanded (LE) and liquid-condensed (LC) states.23,27,28 Plateau indicates the region of DPPC aggregation. The effect of Ca2+ on π-A isotherms of DPPC monolayer shows an increase in the area/molecule with increasing CaCl2 concentration in the LE region. In the condensed region, there is a small decrease in the area/molecule. This clearly indicates that there is a definite interaction of Ca2+ with DPPC at the air/water interface. Our results are quite similar to that reported elsewhere.29 In addition, a small change of height of the hump of
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Figure 1. π-A isotherms: (A) pure DPPC, (B) DPPC spread on 5 mM CaCl2 solution, and (C) DPPC spread on 25 mM CaCl2 solution. Inset shows β-π plots.
the LE-LC phase transition region of DPPC is observed with a higher CaCl2 concentration (25 mM). Binding of the metal ion (Ca2+) to the phosphatidylcholine membrane induces a small conformational change of the phosphatidylcholine headgroup.30 3.2. Compressibility Study. We have performed the surface compressibility analysis of the Langmuir monolayer to specify the phase transition during compression in a better way.31,32 The compressibility coefficient (β) is calculated here by using the following equation:32
β)-
1 δA A δπ
(1)
Interestingly, the LE-LC phase transition peak in the β-π curve indicates the maximum compressibility of the monolayer, revealing maximal intermolecular cooperativeness. The asymmetry of the peak indicates that the phase transition may consist of several steps.29 The β-π curves of pure DPPC and DPPC-Ca2+ with different CaCl2 concentrations (inset of Figure 1) indicate only one phase transition region with a peak at π ) 18 mN/m.32 Importantly, Ca2+ does not alter the phase transition region of pure DPPC.29 An increase in compressibility indicates the increase in intermolecular cooperativeness among DPPC molecules in the presence of Ca2+. 3.3. Penetration of Ca2+ into the DPPC Monolayer. The penetration kinetics of Ca2+ ion in the precompressed DPPC monolayer in the LE (5 mN/m) and LC (35 mN/m) regions at the air/water interface has been studied by injecting a fixed amount of CaCl2 stock solution (to attain 25 mM final concentration) into the water subphase consisting of compressed the DPPC monolayer at a requisite pressure (Figure 2). An increase of π/πPC (π of precompressed DPPC monolayer) with time indicates an interaction of the Ca2+ ions with DPPC, which reaches saturation at about 1 h (trace A in Figure 2). However, in a condensed region, as shown in trace B, a small decrease in π is observed. Thus, the association of the Ca2+ ions in the DPPC monolayer is less in the LC region than in the LE region. The rise in π at πPC ) 5 mN/m may be due to sequential binding of the Ca2+ ions with the carbonyl oxygen, which are present in the hydrophilic headgroup of DPPC.33 At the condensed region, the small decrement of π is due to the reorganization and
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Figure 2. Change in π/πPC with time (t): (A) at πPC ) 5 mN/m and (B) at πPC ) 35 mN/m. Inset shows absolute change of π after injection of 25 mM CaCl2 solution at DPPC monolayer at πPC ) 5 mN/m. The continuous curve in the inset shows the fitted growth curve by eq 2.
domain formation phenomenon of DPPC molecules.23 The penetration of the Ca2+ at πPC ) 35 mN/m is probably less due to the difference in permeability of the respective DPPC monolayer. The penetration kinetics of Ca2+ into the DPPC monolayer at πPC ) 5 mN/m (inset of Figure 2) analyzed using eq 2, is associated with single exponential association mechanism.34,35
πt ) π0 + A1[1 - exp(-t/τ1)]
Figure 3. FE-SEM images: (A) pure DPPC lifted at π ) 5 mN/m (inset, bare glass slide); (B) film lifted at π ) 5 mN/m, DPPC spread on 25 mM CaCl2 solution; (C) pure DPPC lifted at π ) 35 mN/m; and (D) film lifted at π ) 35 mN/m, DPPC spread on 25 mM CaCl2 solution.
(2)
Here πt and π0 are the surface pressures at time t and zero, respectively. Constants A1 and τ1 are the relative contribution and corresponding time constant, respectively. The experimental curve reasonably fits well with the equation, and the residual square correlation coefficient (R2) and the time constant (τ1) are found to be 0.987 and 1176.09 ( 0.41% s, respectively. The agreement of the experimental and the fitted curves shows that the kinetics of the penetration/association of Ca2+ to the DPPC monolayer is adequately described by the single exponential association mechanism method. The larger time constants reveal a slow kinetic process. 3.4. FE-SEM Study. The surface morphology of all of the transferred LB monolayer has been studied by high-resolution FE-SEM. The film (Figure 3A) of pure DPPC at the LE region (π ) 5 mN/m) is not compact, and the formation of domain is about to start. The FE-SEM image (Figure 3B) of the DPPCCa2+ film at the LE region (transferred after 5 min of CaCl2 addition) is also less compact with a few calcium nucleation points. At the condensed state (π ) 35 mN/m), the film of pure DPPC (Figure 3C) is rigid by means of distinct nanometerscale domains/aggregates of DPPC (20-30 nm in diameter) with some holes on the order of a few to 100 nm. These holes originate due to stearic hindrance of the adjacent domains. At the condensed state (π ) 35 mN/m), the film (Figure 3D) of DPPC-Ca2+ is compact, and no domains and holes are observed. In addition, some whitish and nearly spherical dots 6-10 nm in diameter are observed throughout the film. Incorporation of Ca2+ into the DPPC monolayer prevents the domain formation of DPPC, which is also evidenced from the
Figure 4. FE-SEM images. A to C show Ca2+ incorporated DPPC film at π ) 5 (after 2 h at air CaCl2 solution interface) with increasing magnification. D shows the same, but π is maintained at a higher 35 mN/m.
observed shift of the isotherm toward lesser area/molecule and from compressibility by increasing intermolecular cooperativeness. We have additionally studied the growth of these dots with time (Figure 4). Ca2+-incorporated DPPC film at π ) 5 mN/m (after 2 h at air/CaCl2 solution interface) shows the distribution of some single crystals of size ∼100-200 nm (Figure 4A to 4C). Energy-dispersive X-ray spectroscopy (EDX) in FE-SEM (EDX spectrum shown in the Supporting Information) shows traces of calcium, oxygen and carbon, indicating that the crystals are composed of CaCO3. At low pressure, monolayer flexibility is found to be responsible for single crystal formation, whereas at the condensed region, the FE-SEM image of a transferred film (at 35 mN/m and after 2 h at air/CaCl2 solution interface) shows the crystalline aggregated structure of CaCO3 in the micrometer range (Figure 4D).The surface pressure is believed to be a key factor in the single crystal formation. More ordered
Formation of Calcium Carbonate Crystal
Figure 5. HRTEM images: (A) DPPC-Ca2+ film lifted at π ) 35 mN/ m, (B) high-resolution image of A with analysis of an individual dot, and (C) SAED pattern of a dot.
single crystals are found at low surface pressure, which indicates greater dynamic freedom and compressibility than those at high pressure. The Ca2+ may bind with the O- of the DPPC headgroup and binds with a water molecule to form the unstable Ca(OH)2. The atmospheric CO2 at the air/water interface may interact with Ca(OH)2 to form CaCO3. Moreover, assimilation of CO2 at the air/water interface increases the pH of the surrounding medium and shifts the equilibrium toward nucleation.
J. Phys. Chem. C, Vol. 114, No. 18, 2010 8351 3.5. HR-TEM Study. The crystallographic phases of the dots are determined by high-resolution transmission electron microscopy (HRTEM). Figure 5A shows a TEM image of nearly spherical dots 6-10 nm in diameter. The dots are uniformly distributed within the DPPC matrix with regulated spacing, which can be tuned further using applied surface pressure. Relatively larger crystals are also observed, most likely as a result of coalescence of small dots with the aid of surface pressure and the interdot proximity effect.36 The HRTEM image of an individual dot (Figure 5B) shows well-resolved lattice planes with an interplanar distance of 0.201 ( 0.05 nm, which is consistent with the (202) planes of the CaCO3 rhombohedral structure. The rhombohedral structure of the dots is further supported by selected area electron diffraction (SAED), simulated SAED, and simulated powder XRD (see the Supporting Information). The SAED patterns (Figure 5C) obtained from CaCO3 dots show the rhombohedral structure with (202), (113), and (104) diffraction rings, in line with the interplanar distances of 0.201, 0.228, 2.099, and 0.303 nm of the bulk rhombohedral structure (JCPDS 86-0174). The strongest intensity of the 202 reflection indicates a preferred orientation in which the dots are oriented with the diffraction conditions. We have simulated the resultant crystal structure using the bulk parameters a ) 0.49 nm, c ) 1.7 nm, and d202 ) 0.201 nm (Figure 6A). The gray plane denotes the (202) planes when viewed from the 〈110〉 direction. The blue atoms are calcium; red are oxygen; and green are carbon, respectively. Figure 6B and C shows the simulated structure using similar bulk parameters with 3 × 3 × 3 unit cells consisting of 840 atoms. The simulated crystal structure allows us to extract a simulated electron diffraction pattern and X-ray powder diffraction pattern. The simulated diffraction pattern from the (010) projection (using 200KVA and 50 nm aperture) shows the similar diffraction patterns with predominant (202) and (104) intensities (see the Supporting Information); however, the (113) diffraction planes, which are observed in the experimental diffraction pattern, are forbidden. Nonetheless, the existence of the other two planes confirms the CaCO3 rhombohedral structure. The XRD of powder samples of dots matches well to the standard bulk CaCO3 rhombohedral structure with the strongest (104) and (101) reflection intensities (see the Supporting Information). Note the fact that the intensity of the reflections depends on
Figure 6. (A) Simulated resultant crystal structure using the bulk parameters a ) 0.49 nm, c ) 1.7 nm, and d202 ) 0.209 nm; (B, C) simulated structure using similar bulk parameters with 3 × 3 × 3 unit cells consisting of 840 atoms.
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the sample plane orientations and does not always indicates an in-plane direction, which is the growth direction. 4. Conclusion The nucleation and growth of calcite CaCO3 crystals beneath an insoluble Langmuir monolayer of DPPC at the air/CaCl2 mixed water interface at different surface pressures was investigated. The attachment of Ca2+ into the lipid surface was evidenced from a π-t curve, the π-A isotherms, and β-π analysis with a positive association of DPPC with Ca2+ resulting in greater intermolecular cooperativeness. Penetration of Ca2+ into the DPPC monolayer is feasible at a lower surface pressure by the single exponential association method. The surface morphologies and structural analysis revealed formation of wellorganized nanosized CaCO3 crystals having 6-10 nm diameters on the DPPC monolayer by exposure to natural atmosphere. The Ca2+ may bind with the O- of DPPC headgroup or with water molecules to form CaO, which is unstable. At the air/ water interface, atmospheric CO2 may interact with the CaO to form stable CaCO3. The surface pressure allows the monolayer to reorganize to optimize the geometrical and stereo chemical fit and then to accommodate the nucleating or growing crystals. The single crystalline nanodots may be ready to lend a hand in drug delivery as well as in further development of medical sciences. Acknowledgment. We thank DST, the Government of India (Project No.-SR/S2/CMP-0051/2006) for partial financial support. Supporting Information Available: Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Lowenstam, H. A.; Weiner, S. On Biomineralization, Oxford University Press: Oxford, 1989. (2) Mann, S. Biomineralization: Principles and Concepts in Bioinorganic Materials Chemistry, Oxford University Press: Oxford, 2001. (3) Xu, A. W.; Ma, Y. R.; Co¨lfen, H. J. Mater. Chem. 2007, 17, 415. (4) Addadi, L.; Weiner, S. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 4110. (5) Mann, S. Nature 1988, 332, 119. (6) Mann, S. Angew. Chem., Int. Ed. 2000, 39, 3392. (7) Ariga, K.; Hill, J. P.; Lee, M. V.; Vinu, A.; Charvet, R.; Acharya, S. Sci. Technol. AdV. Mater. 2008, 9, 14109.
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