Crystalline Calcium Carbonate Thin Film Formation through Interfacial Growth and Crystallization of Amorphous Microdomains Johannes Pecher,† Patrick Guenoun, and Corinne Chevallard* CEA, IRAMIS, Laboratoire Interdisciplinaire sur l’Organisation Nanome´trique et Supramole´culaire (LIONS), CEA-Saclay, F- 91191 Gif-sur-YVette cedex, France
CRYSTAL GROWTH & DESIGN 2009 VOL. 9, NO. 3 1306–1311
ReceiVed March 6, 2008; ReVised Manuscript ReceiVed NoVember 18, 2008
ABSTRACT: Using a simple interfacial configuration based on poly(acrylic acid) adsorption at an air-water interface, we were able to grow micron-thick crystalline calcium carbonate continuous films. The fine mechanisms of the interaction between the mineral ions and the polyelectrolyte are investigated using combined optical microscopy techniques after synthesizing a fluorescent derivative of the organic polymer. This approach, entirely new in model biomineralization experiments, gives access to the distribution, at the micrometer scale, of the polymer material within the interface, and allows one to derive the time evolution that leads to the mineralized products. In our system, the formation of large partially oriented vaterite crystalline domains at the interface is shown to rely on the early precipitation of amorphous CaCO3 microdomains which subsequently aggregate and crystallize. Introduction Natural mineralized tissues, such as bones, teeth, or mollusk shells, are organic/inorganic composites tailored by specialized cells under mild conditions of pressure and temperature, and grown to fulfill a precise structural or biological function.1,2 Many living organisms exhibit the ability to mold crystalline minerals, like the prominent calcium carbonate, in any desired shape. Mimicking these skills is a challenging issue for materials science, with strong implications for the development of biomaterials in surgery and dentistry, or in ceramics production. The underlying control over CaCO3 precipitation is moreover crucial to prevent the major industrial problem of scaling in fabrics and water pipes. In nature, the existence of transient amorphous precursors is more and more put forward as a key factor of the observed morphological control.3-8 CaCO3 crystalline nanograins, reminiscent of the expected amorphous precursors, have been observed in the calcitic quail eggshell,9 as well as in nacre where the micrometer wide aragonitic tablets of submicron (∼700 nm) thickness are made of assembled nanograins.10,11 Aggregation and crystallization of transiently stabilized amorphous colloidal particles would therefore produce the final biomineral with its original nanostructured shape, far away from the crystal symmetry-dependent shapes. The stabilization of the amorphous phase could be explained by the presence of ionic additives, like magnesium or phosphate ions, as well as by acidic soluble macromolecules adsorbed on the insoluble organic matrix or free in the mineralizing medium. In vitro experiments have consistently shown that nanoparticles of amorphous CaCO3 can be stabilized in bulk by weak polyelectrolytes like poly(acrylic acid) (PAA).12-14 Furthermore, poly(aspartic acid) was shown to induce a liquid-liquid phase separation, thus generating amorphous CaCO3 droplets15 that accumulate on an immersed glass substrate and, owing to their low viscosity, coalesce. The subsequent crystallization of the deposited film produces calcitic tablets similar in shape to the nacreous polygonal tablets. This so-called “polymer-induced liquid precursor” (PILP) process was used elsewhere16 to grow a CaCO3 amorphous film at a fatty acid monolayer located at * To whom correspondence should be addressed. E-mail: corinne.chevallard@ cea.fr. † Current address: Universita¨t Konstanz - Fachbereich Chemie Lehrstuhl fu¨r Chemische Materialwissenschaft, Universita¨tsstr. 10, D-78457 Konstanz, Germany.
an air-water interface, by adding poly(acrylic) acid into the bulk. When no magnesium was present in the medium, the amorphous film transforms into single crystalline patches of calcite. In this paper, we present the conditions for the selective growth of a micrometer-thick film of vaterite, an unstable crystalline polymorph of calcium carbonate at atmospheric pressure and ambient temperature, at an air-water interface. This was achieved by inducing the mineral precipitation in the presence of PAA. The synthesis of a fluorescent PAA-derivative has made it possible to combine confocal fluorescence and birefringence microscopy techniques, and therefore to follow the coupled organization of the organic and inorganic phases during the calcium carbonate precipitation. At the micrometer scale, the mineral film is made of close-packed polygonal tablets, like the mineral layers of nacre, which emerge from the crystallization of amorphous domains initially stabilized by the polyelectrolyte additive. Each tablet develops as a round tablet that becomes polygonal when getting in contact with adjacent ones, similar to the mechanism reported in the formation of Pinctada Margaritifera nacre.17 Experimental Procedures Poly(acrylic acids). All experiments were carried out with the same PAA purchased from Aldrich (Mw ) 2800 g · mol-1; Mw/Mn ) 2.5), except for the synthesis of the fluorescent polymer. In this latter case, a more monodisperse (Mw/Mn ) 1.16 - Polymer Source, Inc., Canada) PAA was used with nearly the same average molecular weight (Mw ) 2500 g · mol-1). PAA was labeled with a fluorescent dye by a carbodiimide coupling reaction18 (see Supporting Information). BODIPY FL hydrazide (D2371, Invitrogen) was chosen as a fluorophore owing to its free amino group that could react with the carboxylate groups of PAA. This fluorescent dye shows an absorption band at 503 nm and an emission band at 510 nm, and could therefore be excited with the 488 nm line of a multiline Argon laser. Precipitation Experiments. Calcium carbonate used to perform mineralization experiments was purchased from Sigma-Aldrich (99+ % purity). Calcium chloride dihydrate from Fluka (purity >99.5%) was employed in control experiments. The so-called “Kitano’s method”19 was used to prepare highly supersaturated CaCO3 solutions. Briefly, carbon dioxide gas was bubbled overnight into a 10 mg · L-1 CaCO3 aqueous suspension under a CO2 pressure of 1.5 atm. The solution was then filtered through a 0.22 µm syringe filter to remove any undissolved particles, and CO2
10.1021/cg800251t CCC: $40.75 2009 American Chemical Society Published on Web 02/06/2009
Interfacial Vaterite Film Formation was bubbled again for 1 h. The solutions were then used immediately. Total Ca2+ concentrations, determined by capillary electrophoresis, were consistently found between 10.0 and 12.4 mM. Capillary electrophoresis is an analytical technique that relies on the different electrophoretic mobilities exhibited by charged species in an electroosmotic flow to identify these charged species, and notably inorganic ions, in solutions and to measure their concentration.20 The precipitation reaction proceeds inversely by CO2 outgassing through the air-solution interface. This causes the precipitation to occur exclusively at the interface. The pH value of the solution, about 6.0 when freshly prepared, increases with time up to about 7.5, due to CO2 escape. Characterization. Fluorescence observations were performed at an excitation wavelength of 488 nm on an Olympus BX61WI upright microscope equipped with a FV1000 confocal head, using either 10× or 40× magnification objectives. Observations were also carried out in transmission mode under crossed polarizers. After the mineralization was complete, the mineralized products were deposited horizontally, with extreme care, on cleaned glass slides. This was done by immersing the slide from one side of the Petri dish and then crossing the interface in a horizontal position to collect the interfacial structures. The collected sample was then dried in air and metallized with a gold-palladium mixture prior to SEM observations on a Hitachi S4500 scanning electron microscope. Alternatively, they could be deposited on (100) silicon wafers for crystallographic characterization on a Siemens D5000 X-ray diffractometer used in a powder configuration (θ-2θ) with CuKR1 radiation. As seen below, in the presence of polyelectrolyte, the interfacial structures were mainly continuous solid-like films, such that no major influence arising from the horizontal deposition procedure was expected.
Results and Discussion The mineralization system used all along this work consists of a 10 mM supersaturated calcium carbonate solution, prepared by Kitano’s method,19 containing different amounts of PAA along with 0.1 wt % of a BODIPY-labeled PAA. Preliminary experiments were performed to ensure that the fluorescent PAA does not influence the mineralized structures with respect to experiments with solely unlabeled polymers. This condition was fulfilled for an amount of 0.1 wt % fluorescent polymer. All experiments were carried out at room temperature, in glass Petri dishes previously cleaned with nitric acid. The samples were prepared by mixing supersaturated calcium carbonate solution with volumes of PAA and fluorescent polymer solutions. The prepared solution was then immediately poured into a glass Petri dish located on the stage of the microscope (Figure 1a-1). Two concentrations of PAA, 40 ppm and 200 ppm, were investigated during this work. Because of CO2 outgassing through the interface (see Experimental Procedures) and to the related increase in the local pH, mineralization took place in the vicinity of the air-solution interface exclusively, over a period of time of about 48 h. As a reference experiment, a supersaturated 10 mM calcium carbonate solution without polymeric additive was kept in a covered glass Petri dish for 20 h, and showed typical rhombohedral crystals of calcite, the most stable CaCO3 polymorph, aggregated at the air-solution interface into an irregular network (Figure 1a-2). Samples containing 40 ppm and 200 ppm of PAA dissolved in the aqueous subphase exhibited totally different mineralization products. After several hours (∼10 h), extended crystalline domains showing a Maltese-cross birefringence pattern, typical of a 2D spherulitic growth, could be observed at the surface of the solution for both concentrations (Figure 1b-1,c-1). In the presence of 40 ppm PAA, these domains adopt a round shape with a diameter size ranging from about 100 to 200 µm (Figure 1b-1) and form a discontinuous pattern at the air-water interface with no visible evolution beyond about the 20th hour. SEM micrographs of this horizontally deposited
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pattern show the same round domains and reveal holes in their structure (Figure 2a-1). Some of these domains are curved, which makes it possible to view both sides simultaneously and to estimate their thickness (Figure 2a-2). As a matter of fact, each domain is very akin to a film island since its thickness is about 1 µm, whereas its diameter is about 100 µm. In the presence of 200 ppm PAA, domains are no more scattered at the air-water interface like islands, but rather form a continuous crystalline film made of polygonal domains (Figure 1c-1). However, the domains exhibit characteristics (cross-type birefringence, thickness) similar to those formed in the 40 ppm case. These packed polygonal tablets show the typical thickness and shape of nacreous tablets. However, they are not single crystals of aragonite but rather polycrystalline vaterite domains which grew radially and got their final facetted shape by contacting each other. As seen in Figure 1b-1 and 1c-1, the crystalline islands or film (white arrows) observed for the 40 ppm and 200 ppm concentrations respectively develop along with other birefringent thicker crystalline structures (green arrows). However, these additional mineralization products represent a smaller percentage of the birefringent material at 200 ppm than at 40 ppm. A first major outcome of our observations is that increasing the polyelectrolyte concentration in the bulk promotes the growth of an extended mineral film at the air-water interface over the development of these additional structures. The features and conditions of formation of the above thicker structures will be considered in a future publication. The observed radial distribution of birefringence of the micrometer-thick crystalline films is indicative of a uniform averaged orientation of the polycrystals in the radial direction and of a continuous change in the azimuthal direction (Figure 1b-1). In order to identify the polymorphic type of the crystal, X-ray powder diffractograms were recorded on the horizontally collected interfacial structures. For both 40 ppm and 200 ppm concentrations, the diffractogram shows peaks that correspond to two anhydrous polymorphs of the crystalline calcium carbonate, namely, calcite and vaterite. However, calcite peaks are much more intense in the 40 ppm sample than in the 200 ppm sample. Considering that the quantitative ratio of the additional crystalline structures to the micrometer-thick film is much higher at 40 ppm than at 200 ppm, and assuming that the thin film structure is similar for the two systems, X-ray data therefore indicate that the calcite peaks originate from the additional thicker structures, whereas the thin crystalline CaCO3 films formed both at 200 ppm and 40 ppm consist of vaterite polycrystals. Moreover, whereas all calcite peaks are present, some of the vaterite peaks do not appear in the diffractograms. Thus, the (112) peak of vaterite, referred to as the 100% intensity peak in X-ray data tables, is not present in both cases, while the (110) and (300) peaks are clearly identified. The lack of several vaterite peaks indicates a partial orientation of the vaterite crystallites within the spherulitic domains in both samples. More precisely, either the (110) face or the (300) face of the crystallites is parallel to the air-water interface, which amounts to saying that the c-axis (001) lies everywhere parallel to the interface. Vaterite is an unstable CaCO3 polymorph which usually transforms into calcite very fast under standard conditions. While vaterite stabilization in bulk using additives has been achieved by many groups and, in a few cases, leads to stabilization over long periods of time,21-23 there are, to our knowledge, only three reports in the literature of the formation of continuous pure vaterite thin films,24-26 while in other reports only mixed
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Figure 1. (a1) Experimental setup used in the mineralization experiments. (a2) Typical rhombohedral calcite crystals nucleated at the air-solution interface after 20 h (in situ brightfield micrograph). (b1) In situ optical micrograph, under crossed polarizers, of the mineralized products formed at the air-water interface in the presence of 40 ppm PAA (t ) 22 h): white arrows point at surface film islands, and green arrows point at additional 3D structures (b2) X-ray diffractogram (Cu KR1 radiation) of the horizontally collected mineralized structures. c stands for calcite diffraction peaks, and v is for vaterite peaks. The peak located at 2θ ) 33° is due to the (100) silicon wafer used to collect the crystals. (c1, c2) Mineralized structures formed at the air-water interface in the presence of 200 ppm PAA (t ) 49 h): (c1) in situ optical micrograph under crossed polarizers of the continuous crystalline film; (c2) X-ray diffractogram.
(calcite/vaterite or aragonite/vaterite) or discontinuous vaterite thin films could be formed.15,27-29 Wada and coauthors26 grew a vaterite film on a chitosan membrane in the presence of PAA at basic pH () 9.2) using a multistep procedure. Very interestingly, the X-ray diffraction pattern of the vaterite film formed under these conditions looks quite similar to the one shown in Figure 1c. In particular, both exhibit two (110) and (300) main peaks, which indicates a partial orientation of the vaterite crystallites with respect to the chitosan membrane or to the air-water interface respectively. Our system, based on CaCO3 precipitation at an air-solution interface in the presence of 200 ppm PAA, therefore defines a simple, one-step procedure to produce micron-thick vaterite films without the need to prepare an organic template.
Whereas the birefringence signal under crossed polarizers reveals the presence of any crystalline material at the interface (Figure 2b-1), the interfacial polyelectrolyte distribution can be visualized by confocal fluorescence microscopy (Figure 2b-2). We then used combined confocal fluorescence and polarization imaging to unravel the interplay between the organic and inorganic phases during the interfacial precipitation of CaCO3. Using this strategy, we could evidence the initial precipitation of an amorphous phase of calcium carbonate. This amorphous phase takes the shape of a fluorescent film made of close packed contiguous domains with no associated birefringence, visible in Figure 2b-2. This conclusion can be drawn from a comparative experiment where the free surface of a CaCl2 solution was imaged under similar PAA concentration and pH values (40
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Figure 2. (a1-a2) SEM images of vaterite islands. (b) In situ photographs of vaterite island domains formed at the liquid-air interface with [CaCO3] ) 10 mM, [PAA] ) 40 ppm, mineralization time ) 48 h: (b1) under crossed polarizers; (b2) using fluorescence confocal microscope.
ppm PAA, pH ∼ 6) but in the absence of added carbonate ions: in this case, the noncrystalline fluorescent structures do not form, but rather the interface becomes uniformly fluorescent owing to polyelectrolyte adsorption at the air-water interface. Therefore, the fluorescent film domains in Figure 2b-2 very likely coincide with an amorphous CaCO3 phase that first precipitates all over the air-water interface under the influence of the polyelectrolyte additive. The subsequent transformation of this amorphous phase into the crystalline polymorph vaterite gives rise to the observed polycrystalline islands (see Figure 2b-1). To get more details on the precipitation mechanisms, the temporal evolution of a mineralizing system consisting of a 10 mM supersaturated calcium carbonate solution with 40 ppm of PAA was studied using simultaneously confocal fluorescence, crossed polarizers, and phase contrast modes. As the surface was absolutely rigid and exhibited no motion, the same zone could be imaged for 12 h. Figure 3 presents confocal fluorescence, phase contrast, and birefringence micrographs taken at different times. Figure 3a shows an air-water interface at short times (∼17 mn) which exhibits a highly heterogeneous fluorescence. Later, small highly fluorescent micron-sized domains form at the interface (typical size of about 20 µm as shown in Figure 3b). These microdomains are amorphous CaCO3 domains resulting from the polyelectrolyte adsorption at the interface in the presence of carbonate ions, and look like the interfacial counterpart of the PILP droplets formed during the bulk liquid-liquid phase separation reported in ref 15. They look monodisperse in size and are evenly distributed over the surface of the solution. They grow laterally with time (Figure 3b-d) until they seem to coalesce into a continuous film (Figure 3e, similar to Figure 2b-2). The fluorescence signal shows some spatial heterogeneity, with a higher intensity at the periphery of the microdomains, likely due to PAA accumulation. In these early stages (from (a) to (e)), no birefringence can be detected, whereas the phase contrast mode reveals the same surface features as fluorescence, but with lack of contrast.
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The first crystalline islands are observed under crossed polarizers after about 9 h (g). These crystalline areas grow laterally with time as demonstrated by phase contrast microscopy, and birefringence observations (g-j). When regarding the corresponding fluorescence micrographs (g-i), the appearance of the thin crystalline domains goes along with a decrease in the fluorescence intensity, although weakly detectable in the early stage of crystallization (h). As PAA is known to inhibit CaCO3 crystallization, the ejection of this polyelectrolyte is indeed expected to occur during the amorphous-to-crystal transformation of the interfacial film. The crystalline island formation mainly occurs between 9 and 11 h after the start of the experiment. As previously demonstrated, these crystalline islands are micrometer-thick film islands (see Figure 2a-2). At the end of the island growth period (after about 10.5 h, see (i-j)), 3D structures become visible in the form of highly fluorescent points which form below amorphous or crystalline interfacial structures equally, as proven by SEM micrographs (not shown). One can note a renewed fluorescence intensity of the crystalline 2D domains in image (j). This likely corresponds to the readsorption of some PAA on the calcium rich (300) and (110) faces of vaterite exposed to the subphase. In the case of a supersaturated 10 mM CaCO3 solution containing 200 ppm PAA, micrographs taken during the mineralization process at the interface pointed up the same generic mechanism. Figure 4a,b is two snapshots showing the same interface location at time t ) 64 h from the start of the experiment, imaged simultaneously in confocal fluorescence mode and under crossed polarizers. A continuous film of spherulitic vaterite is visible in the upper part of the pictures, whereas the lower part shows micron-sized (∼20 µm) disconnected interfacial domains, some of them being birefringent (regions II and III), others being amorphous (region I). Figure 4c, which is a fluorescence image of the interface, at a different location, at time t ) 105 mn from the start of the experiment when no birefringence is detected, actually indicates that the amorphous structures formed at the interface are heterogeneous: although large film-like patches prevail at the interface, they locally coexist with micron-sized domains. Figure 4b can therefore be interpreted as the result of the amorphous-to-crystal transformation of different amorphous structures whose shape has been “frozen” at the early stages of the precipitation mechanism. Some microdomains, although similar to those observed in the 40 ppm sample (see Figure 3b), may have been unable to coalesce with the continuous amorphous film and would only accumulate along it (see Figure 4c). Figure 4b shows that microdomains neighboring the vaterite film crystallize preferably to more distant domains, suggesting an influence of the already crystalline film on the surrounding microdomains. Then, assuming that the crystallization of the microdomains is triggered by the crystalline film, one can identify again in Figure 4a,b the different stages of precipitation, similar to those characterized from the 40 ppm sample kinetic study. In region I, that is far away from the crystalline film, the microdomains appear fluorescent with no birefringence. As seen previously, these microdomains are glassy CaCO3 domains, rich in PAA and represent the first stage of precipitation. When getting closer to the film, in region II, the appearance of a birefringence of the microdomains, visible in Figure 4b, exactly coincides with a partial loss of their fluorescence visible in Figure 4a. Lastly, in the immediate vicinity of the main vaterite film (region III), the crystalline
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Figure 3. All shown micrographs (200 × 200 µm2) correspond to the same sample containing 40 ppm PAA. The same location of the liquid-air interface was imaged at different times of mineralization using different optical techniques (confocal fluorescence, birefringence, and phase contrast imaging): (a) 17 min, (b) 1 h 32 min, (c) 2 h 21 min, (d) 3 h 25 min, (e) 5 h 09 min, (f) 6 h 59 min, (g) 9 h 05 min, (h) 9 h 52 min, (i) 10 h 41 min, (j) 12 h 34 min. The black arrow indicates the location of the dust which initiates the film crystallization; the white arrow shows the region of lower fluorescence intensity under crystallization.
microdomains regain a marked fluorescence, which could correspond to the readsorption of PAA on the crystallographic faces (300) and (110) exposed to the solution.
emphasizes the paramount importance of controlling the amorphous phase precipitation.
Therefore, the common features of the mineralization experiments described above are (1) the formation at an early stage of CaCO3 glassy structures that cover the whole interface; (2) the spherulitic growth of vaterite which corresponds to the amorphous-to-crystal transformation of these structures with no associated modification of their boundaries at the micrometer scale. Interestingly, the difference in bulk polymer concentrations result in different amorphous interfacial structures: whereas the amorphous phase forms a loose continuous film made of interconnected microdomains (see Figure 2b-2) in the 40 ppm sample, it forms dense films, likely resulting from the coalescence of microdomains, as well as isolated microdomains in the 200 ppm sample (Figure 4c). This difference, in turn, is reflected in different crystalline structure morphologies. This
Conclusion The mineralization model system presented in this study catches several typical conditions of nacre formation owing to the presence of an acidic polymer akin to the carboxylic groups very frequently found in shell proteins. The structure of the crystalline CaCO3 continuous films, thereby selectively produced using PAA, consists of densely packed polygonal tablets of polycrystalline vaterite whith submicron thickness. Thus, this simple experimental setup leads to some morphological control on the precipitated mineral, a typical feature of biologically controlled mineralization. Moreover, the mineral is precipitated out of an unstable polymorph of CaCO3, namely vaterite, instead of the most stable calcite polymorph. This is a sign of a possible polymorph selection, notably at work in nacre formation where the CaCO3 aragonite polymorph is exclusively produced. The
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along these lines should provide more resolved pictures of the bioinspired formation of minerals. Acknowledgment. This work was supported by the European Master “Complex Condensed Materials and Soft Matter” (EMASCO-COSOM) and by the French research program ANR-07-NANO-061-01. Supporting Information Available:
Experimental procedures for PAA fluorescent labeling, purification, and characterization of the product are available free of charge via the Internet at http://pubs.acs.org. References
Figure 4. (a) Confocal fluorescence and (b) birefringence snapshots, at time t ) 64 h, of the same location at the liquid-air interface of a supersaturated solution containing 200 ppm PAA. (c) Confocal fluorescence at time t ) 105 mn of the same sample but at a different location.
precipitation of calcium carbonate at an air-water interface in the presence of polyelectrolyte is certainly one of the simplest biomimetic systems that accounts for the main ingredients of nacre biomineralization. Using this system, films of calcium carbonate can be produced without the need for the organic templating provided by Langmuir monolayers or polymeric substrates, merely by inducing the surface precipitation of a supersaturated solution containing 200 ppm of poly(acrylic acid). The mineral film may then easily be deposited on a positively charged substrate for further use. However, to go deeper in the understanding of the biomineralization processes, the additional influence on the mineral precipitation of an organic layer of peptides, which mimics the organic matrix of nacre, has certainly to be considered. Such an issue is currently under investigation in our laboratory, where the interfacial self-assembly of amyloid peptides has recently been shown to produce crystalline β-sheet layers.30 To our knowledge, this publication reports the first direct visualization by combined optical microscopy techniques of the organic/mineral interplay during a biomimetic mineralization experiment. The simultaneous observation of the organic and inorganic components enabled the detailed investigation, at the micrometer scale, of the polymer-driven morphogenesis of CaCO3 mineralized structures at an air-water interface. In particular, the detectable precipitation of specific amorphous CaCO3 structures at the air-water interface is shown to exert an essential influence on the following formation of vaterite crystals. In view of the results already obtained, and given that the spatial resolution in confocal optical microscopy can be increased up to about 200 nm, we believe that further work
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