Synthetic Seashells: Biomimetic Mineral Nucleation at a Langmuir Monolayer E. DiMasi1, V.M. Patel2, S. Munisamy2, M. Olszta2, and L.B. Gower2 1Brookhaven National Laboratory 2University of Florida Biogenic minerals, formed by living organisms, registry between the template and the final crystal is have properties quite different from those of their inor- called into question – the template may still influence ganic counterparts. Mollusk shells are exemplary crystallization, but the mechanisms for crystallization biominerals. They are laminates, composed of calcium from a dilute solution are likely to be quite different from carbonate, layered with an organic matrix. These ma- those of crystallization from a dense viscous or solid terials are structured in a hierarchical manner, in which phase. Neither optical microscopy nor electron diffrac- micron-scale mineral crystallites of controlled shape and tion can provide the necessary structural information orientation are arranged into the larger pattern. Ther- to clarify this problem. modynamically stable and unstable mineral forms can In-situ synchrotron x-ray scattering studies can fill coexist within the same organism, such as for the red this gap and shed new light on the mechanisms of abalone (Haliotis rufescens) where the exterior of the biomineralization. In this article we describe recent ex- shell is calcite, but the nacre (mother-of-pearl) layer is periments on biomimetic calcium carbonate nucleation. composed of crystals of aragonite, a less stable poly- Our model system consists of a liquid subphase and a morph of calcium carbonate. Nacre is twenty times surfactant monolayer, and provides several advantages. stronger, in terms of both fracture toughness and spe- First, the monolayer template’s surface charge and lat- cific flexural strength, than inorganic calcium carbon- tice spacing may be tuned through careful choice of ate, due to its laminated structure [1]. Such structural the organic surfactant, and by the application of sur- organization on nanometer to micron length scales in face pressure [4]. Similarly, the ingredients of the synthetic materials would be highly desirable, but this subphase can be controlled, and may include the nucle- goal has not yet been achieved. ating species along with additional metals or organic What will it take to “grow” biomimetic (that is, syn- molecules thought to influence crystallization. Most thetic but bio-inspired) composites? It is clear that the importantly, in-situ x-ray scattering allows us to deter- routes to biomineralization are very diverse. For ex- mine the structure of the mineral from its inception as a ample, certain crustaceans, sponges, and other organ- collection of calcium ions at a monolayer interface, to isms produce amorphous CaCO3 [2]; abalone shell is various bulk phases including macroscopic crystals as microcrystalline; and sea urchin spines are millimeter- well as amorphous thin films. We make use of two long “single crystals” with exceptional alignment across complementary techniques. X-ray reflectivity is a sen- domain boundaries [3]. The complex morphology of sitive probe of the density profile along the surface nor- biogenic minerals is controlled by two important aspects mal direction. It is essentially a measurement of inter- of their environment during mineralization: the soluble ference between x-rays reflecting from boundaries par- chemical species present, which affect the rate of crystal allel to the surface: in our case, the air-monolayer and nucleation and growth, and can also stabilize different monolayer-mineral or monolayer-water interfaces are crystal faces; and an organic insoluble substrate, which identified and the density of each region can be mod- can provide a template for controlled nucleation of spe- eled and fit to the data [5]. In-plane diffraction mea- cific crystallographic phase or orientation, and acts as surements are also conducted, with the x-ray beam at a boundary surface for controlling crystal size and grazing incidence to the surface, so that the near-sur- shape. The relative importance of the template versus face region rather than the bulk is probed. This way, the kinetics is not understood in a general way for the surfactant molecule spacing in the plane as well as biomineralizing systems. One reason for this is that up the crystallinity of the underlying mineral can be deter- to now, no in-situ probes on atomic length scales have mined. The present work was performed using the been available. This means that even when an organic Harvard-BNL liquid surface spectrometer at NSLS material forming the boundary surface of the biomineral beamline X22B. appears to have a good registry with the atomic posi- Our experimental conditions are illustrated in the tions of the crystal faces, it has been very difficult to top panel of Figure 1. In all cases, the surfactant is confirm that the mineral is in fact crystalline at early arachidic acid (CH3[CH2]18COOH): when spread from growth times. If mineralization proceeds first via an chloroform solution onto an aqueous subphase and amorphous precursor phase, the significance of atomic compressed in a Langmuir trough, the molecules stand NSLS Activity Report 2001 2 - 138 up to form a monolayer at the air-water inter- face. In Scheme (a), the subphase consists of a diluted calcium bicarbonate solution which is acquired by bubbling CO2 through a suspension of calcite crystals [6]. The deprotonated -COOH headgroups are ex- pected to attract a layer of calcium ions (shown as red balls) at the surface. As CO2 gas escapes from the liquid, the calcium carbonate supersaturation is raised and mineralization proceeds. This system was previously shown to favor the nucleation of oriented vaterite crystals at the surface, as opposed to the calcite that forms in the ab- sence of a monolayer [7]. Schemes (b) and (c) incorporate acidic macromolecules and metal ions, as found in biological environ- ments. In scheme (b), poly(acrylic acid) is added to the supersaturated calcium bicar- bonate solution. CO2 gas escape drives mineralization just as in scheme (a). The polymer (shown as scribbles in the figure) retards calcite crystallization and forces mineralization to proceed via a hydrated amorphous phase [8]. In scheme (c), car- bonate supersaturation is controlled in a 2- different way: here, CO3 species are sup- plied by pumping an ammonium carbonate solution into a solution containing Ca2+, along with Mg2+ and poly(aspartic acid) as inhibitors. Under some conditions this recipe forces mineralization through a novel liquid precursor phase discovered very re- cently [9]. This polymer-induced liquid-pre- cursor (PILP) process was previously found to form macroscopic amorphous films at stearic acid monolayers on water, and sub- sequently to crystallize into calcite. An opti- cal micrograph through crossed polarizers, showing a spongy amorphous film along Figure 1. Top: recipes for mineralization, beginning with a surfactant with precursor droplets, is shown in Figure monolayer (arachidic acid, CH3[CH2]18COOH) on an aqueous 2. When the precursor is in the liquid form, subphase. (a) Supersaturated calcium bicarbonate solution diluted it can flow into cavities of arbitrary shape, 1:1 with water to produce an undersaturated solution. Carbon diox- and upon solidification, the crystalline cal- ide gas escape raises calcium carbonate saturation and promotes cite then retains the morphology of the mineralization. (b) Supersaturated calcium bicarbonate subphase with molding space. This process suggests a polyacrylic acid. (c) Calcium and magnesium cations in solution, car- bonate solution pumped into subphase. Bottom: possible outcomes simple mechanism for “molding” crystalline for mineralization. (d) Crystallization controlled directly by atomic biogenic minerals to obtain the curved registry with monolayer template. (e) Amorphous mineral precursor, shapes so often observed in nature, and collected at charged boundary surface. (f) Mineral precursor nucle- for this reason, understanding the liquid ates without interacting with monolayer. Red balls, green balls, bent precursor phase is of special interest. Min- molecules, triangular molecules, and black scribbles represent Ca eralization therefore proceeds through di- cations, Mg cations, water molecules, carbonate groups, and soluble verse routes, which we summarize sche- polymers respectively. matically in the bottom panel of Figure 1. Figure 1(d) shows the template-driven ex- 2 - 139 Science Highlights treme proposed in the literature, where crystal faces region, shown by the corresponding model in Figure align directly at the monolayer template. Figure 1(e) 4(b). This may be explained by Ca2+ ions collecting at describes an amorphous mineral layer (whether liquid the charged headgroups. After a twelve hour interval, or solid), that simply collects at the charged boundary the reflectivity is essentially the same, except for being surface and may subsequently crystallize. We must also much lower near q=0. This is simply due to homoge- be prepared for the situation depicted in Figure 1(f), in neous nucleation of macroscopic calcite crystals from which the mineral phase ignores the “template” and solution, which clutter the surface and interrupt the nucleates on its own. beam at low incident angles. Grazing incidence diffrac- X-ray reflectivity is the most effective way of distin- tion in this case was consistent with hexagonally guishing the structures described above. A reflectivity packed, untilted