Tailoring Calcite Growth through an Amorphous Precursor in a

Article Cite This: Cryst. Growth Des. 2019, 19, 3192−3205

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Tailoring Calcite Growth through an Amorphous Precursor in a Hydrogel Environment Josue A. Lopez-Berganza,§ Siyu Chen,‡ and Rosa M. Espinosa-Marzal*,§,‡ §

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Civil and Environmental Engineering, University of Illinois Urbana−Champaign, 205 North Mathews Avenue, Urbana, Illinois 61801, United States ‡ Materials Science and Engineering, University of Illinois Urbana−Champaign, 1304 West Green Street, Urbana, Illinois 61801, United States S Supporting Information *

ABSTRACT: The precipitation of calcium carbonate in hydrogel-like environments is used by certain living organisms to build functional mineral−organic composite structures. Here, we investigate a pathway for calcium carbonate mineralization in agarose hydrogels with a wide range of polymer networks. The experimental investigation demonstrates that the formation of amorphous calcium carbonate (ACC) throughout the agarose hydrogels is a diffusion-limited process, and therefore, it is affected by the supersaturation of the solution and by the hydrogel network. In contrast, the inclusion of the polymer into the calcite crystals and their morphology as well as the rate of crystal growth are controlled by the amorphous precursor, and thereby, they are quite unaffected by the initial supersaturation. The nucleation rate of calcite in agarose is sufficiently high to hinder ion diffusion limiting the calcite growth rate, so that a uniform mineralization takes place in the hydrogel, in the absence of concentration gradients. This work demonstrates that the precipitation of ACC affords a tight control of calcium carbonate mineralization in the hydrogel over a wide range of calcium carbonate concentrations and hydrogel microstructures. The results of this work not only reveal an important mechanism underlying (bio)mineralization, but they can also inspire a new avenue to craft biomimetic materials with a high degree of precision. phases.4−8 ACC particles may either dissolve and recrystallize or undergo a solid-state transformation into crystalline polymorphs. The growth of these minerals is often tailored within a hydrogel-like environmenta natural or synthetic polymer matrix holding a large amount of watercontaining soluble organic matter.9 In nacre for example, silk-like proteins create a hydrogel-like environment and act in conjunction with acidic proteins to control the morphology of the nascent crystal.10 The high level of control that can be achieved through the diffusivity, chemical functionalities, and ionic environment in a hydrogel allows systematic evaluation of different influencing factors on mineralization.11 As a result, a number of combinations of hydrogels and minerals have been examined as a way to shed light on the physicochemical mechanisms

1. INTRODUCTION Calcium carbonate, the most common among biogenic minerals,1 forms the basis of numerous skeletal components in marine organisms.2 These organisms exploit several physical and chemical interactions between calcium carbonate and organic matter to guide the precipitation process through thermodynamic and kinetic pathways that yield visually stunning and highly functional composites. Within these hierarchically organized materials, calcium carbonate exists either as an anhydrous crystalline polymorph (calcite, vaterite, or aragonite) or as various amorphous phases (ACC).3,4 While earlier studies had shown that marine organisms favor crystalline calcium carbonate because it provides high strength, and thus, protection, the crucial role of ACC as a precursor to these crystalline structures has drawn tremendous scrutiny in recent years. Indeed, it is now widely reported that organic or inorganic stabilizers open pathways to precipitate a number of different prenucleation species of calcium carbonate that may transform into ACC nanoparticles or directly into crystalline © 2019 American Chemical Society

Received: January 14, 2019 Revised: May 8, 2019 Published: May 9, 2019 3192

DOI: 10.1021/acs.cgd.9b00062 Cryst. Growth Des. 2019, 19, 3192−3205

Crystal Growth & Design

Article

underlying biomineralization.12,13 In the case of calcium carbonate, specific chemical moieties of the hydrogel’s polymer can be used to promote either calcite,14 vaterite,15 or aragonite16 crystallization. The chemistry of the hydrogel network can also set the supersaturation threshold for nucleation and thereby the nucleation rate.9 As a result, the size and quality of the crystals can be also modulated.17 The interactions of the growing crystal with the hydrogel network can be tuned to favor incorporation of the organic phase into the crystalline body.18 Systematic studies of calcium carbonate precipitation in agarose hydrogels through the gas diffusion method showed that a competition between the growth rate and the strength of the hydrogel network dictates whether the hydrogel network is embedded within calcite or pushed away by the mineral.12 Fast growth rates were determined to favor growth of calcite around the fibers due to the reduced time for mass transport to the mineral−organic interface. Strong hydrogel networks were able to resist the crystallization pressure exerted by the crystal, forcing growth around the fibers and enhancing agarose incorporation.12 The same mechanisms dictating the hydrogel embedding and their effect on the crystal structure have also been reported in double-diffusion crystallization experiments where calcite was precipitated in agarose and gelatin hydrogels at much higher supersaturations (0.5 M CaCl2 and 0.5 M Na2CO3) and polymer concentrations (1 and 10 wt %) compared to previous works.19−21 Tomographic imaging of these systems showed that the organic network remained intact within large volumes of calcite and that convex crystalline faces developed at the mineral−organic interface in order to accommodate the curvature around the fibers.22 These studies highlighted the important role of the physical and chemical properties of the hydrogel network in dictating the crystal growth, and by extension, the microstructure of the resulting mineral composite. The inclusion of organic and inorganic matter within the crystalline lattice has been reported to affect morphological,19−21,23 optical,24,25 magnetic,25,26 and mechanical properties of the mineral.27 In this work, we describe a pathway to tightly control mineralization in agarose hydrogels over a wide range of calcium carbonate concentrations through the formation of an amorphous precursor. Agarose is a neutral linear polysaccharide isolated from red algae with repeating units of 1,3 β-Dgalactopyranose−3,6 anhydro-α-L-galactopyranose. As a physically cross-linked hydrogel, it relies on hydrogen bonds to form a three-dimensional network, consisting of rod-like fibers with a characteristic mesh size and fiber thickness.28 Agarose hydrogels are routinely used in crystal growth studies; they are considered to weakly interact with the ionic environment due to the lack of charged moieties that would lead to calcium or carbonate binding.9 Here, agarose hydrogels with varying concentration of agarose were prepared to modulate the mesh size and fiber thickness, which led to a variable ion diffusivity and hydrogel strength.29,30 Microscopy, spectrophotometry, Raman spectroscopy, and thermogravimetry were combined to shed light on the mineralization pathway within the hydrogel environment. The agarose network is found to modulate, through ion diffusion, the formation of amorphous calcium carbonate within the hydrogel, as a precursor to calcite, over a wide range of calcium carbonate and agarose concentrations. This amorphous precursor phase is found to tightly control the growth rate of calcite, the crystal morphology, and the incorporation of the organic phase into the crystal and to

afford a uniform mineralization of the hydrogel, independently of the bulk concentration and the hydrogel network.

2. MATERIALS AND METHODS The following chemicals were used without further purification: agarose (Sigma-Aldrich, 120 000 g/mol, molecular biology grade (A9539), Low EEO, ≤0.15% sulfate anions), calcium chloride dihydrate (Sigma-Aldrich, ≥99%), and sodium carbonate (SigmaAldrich, ≥99%). 2.1. Sample Preparation. Calcium chloride (CaCl2) was dissolved in deionized water (DI) at the selected concentrations of 0 (reference), 5, 15, 50, 100, and 200 mM during continuous stirring and filtered with a 0.2 μm Nylon filter (Fisherbrand, USA). Agarose was added to the CaCl2 solutions at the concentrations of 0.5, 1, 2, or 5 wt %. The polymer solution was heated to 90 °C until agarose was fully dissolved in a sealed container to avoid evaporation, and then, it was pipetted into tissue culture dishes (diameter: 35 mm, thickness: 3 mm, TPP, Switzerland) and covered with a glass coverslip to form hydrogels with a thickness of ∼1 mm. After 30 min of gelation at room temperature, the hydrogels were transferred to larger Petri dishes (diameter: 100 mm, height: 15 mm, Fisherbrand, USA) where mineralization was induced upon addition of an equimolar sodium carbonate (Na2CO3) solution with a volume ratio of 20:1 with respect to the CaCl2 solution. Reference mineralization experiments (without hydrogels) were conducted by mixing 1 mL of equimolar CaCl2 and Na2 CO3 solutions in a tissue culture dish at the selected concentrations. 2.2. Mineralization Kinetics of the Hydrogels. Immediately after the Na2CO3 solution was introduced into the fluid cell, the sample was illuminated with a fiber optic light source (Techniquip, FOI-150, USA), and the transmitted light through the hydrogel was recorded by a spectrophotometer (Ocean Optics, USB+2000, USA) during mineralization. Similar spectrophotometric measurements were extensively used in our previous work31 to quantify the kinetics of mineralization of calcium carbonate in thin polyelectrolyte films. In brief, the time-resolved attenuation of the light by the sample (also called, the optical density) Aλ = − log10

Iλ − Dλ I0 − Dλ

(1)

is measured at the wavelength λ = 530 nm, Iλ and I0 being the transmitted light intensity through the hydrogel and the incident light, respectively, and Dλ the background intensity, which is measured before each experiment. The attenuation is originated by the absorbance (αλ) and the scattering (τλ) of light by the sample composed of the hydrogel and the mineral:

Aλ = aλ + τλ = c(ελ + σλ)d

(2)

where c is the number density of the absorbant (per m ), ελ the absorptivity coefficient (in m2), which depends on the electronic properties of the sample, σλ the scattering cross-section (in m2), and d the thickness of the hydrogel (1 mm). The first term is known as the Beer−Lambert law of spectroscopy and implies that absorbance increases with concentration of the absorbant according to the absorptivity coefficient at the selected wavelength.32 The scattering depends on geometry, size, and refractive index (np) of the scattering particles. Since the particles responsible for the light scattering are mainly the ACC nanoparticles, as demonstrated later, whose size is smaller than the wavelength of light, the Rayleigh theory can be used to estimate the scattering contribution to the attenuation.33 Assuming that all the particles are of the same size, this yields following expression for the scattering cross-section: 3

σλ ≈

4 8π ij 2πnmed yz 6ijj m2 − 1 yzz jj zz a jj 2 z 3 k λ { jk m + 2 zz{

2

(3)

α being the particle size, nmed and np the refractive index of the medium and of the particles, respectively, and m = np/nmed. 3193



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°C for 45 min to digest the agarose network. The crystals were then filtered for further characterization. 2.4. Critical Drying of Hydrogels. At the end of the mineralization process, water in the hydrogels was exchanged with ethanol by immersing them in a series of aqueous solutions with increasing ethanol concentration (30, 50, 70, 90, and 100%), each for 30 min, and finally the hydrogels were immersed in pure ethanol for 24 h. After complete exchange of water by ethanol, the hydrogels were dried in a critical point dryer (Tousimis, Autosamdri 931, USA) with liquid CO2 as the exchange liquid. The samples were held at the supercritical point of CO2 for 2 min prior to evacuation. The hydrogels were also critically dried after 3 min of mineralization; these hydrogel samples were directly immersed in 100% ethanol to quench the reaction and preserve the precursor phase. 2.5. Mineral Identification. Raman spectroscopy, IR-spectroscopy, X-ray powder diffraction (XRD), scanning electron microscopy (SEM), and dynamic light scattering (DLS) were used to characterize the minerals precipitated in the hydrogels after 3 and 6 min of mineralization and at the end of the mineralization process. To quench the mineralization after 3 and 6 min, the hydrogels were immersed in ethanol, and then, critically dried. In the reference experiments in the absence of the hydrogel, the precipitates were filtered 3 min after mixing the two solutions, and the solids were filtered, dried with ethanol, and stored in a vacuum for further characterization. Raman spectroscopy (Nanophoton, Raman-11, Japan) was used with a laser of 60 W (532 nm) and irradiation time of 20 s. The spectra were collected over the range of 60−2000 cm−1. Attenuated total reflectance infrared spectroscopy (PerkinElmer, Frontier, USA and Pike Technologies, GladiATR, USA) was used to collect IR spectra of the critically dried mineralized hydrogels in the range of 600−4000 cm−1. Characterization of the crystallinity of the mineral phase (a) in the critically dried hydrogels, (b) after being isolated from the hydrogels, and (c) in the control measurements in the absence of hydrogels, was conducted by XRD (Siemens-Bruker, D5000, Cu-K alpha source, 0.15418 nm, USA). XRD diffractograms were collected between 2θ angles of 20 and 70° at a scan speed of 5° min−1. Both the isolated precipitated minerals and the critically dried hydrogels at two different mineralization stages (after 3 min and after mineralization was concluded) were also imaged by SEM. The samples were fixed with carbon tape and sputtered with a gold palladium (Au/Pd) for 30 s for SEM imaging under high vacuum (Hitachi, S4700, Japan). To determine the size distribution of the amorphous nanoparticles, DLS measurements (Malvern, ZS90, United Kingdom) were performed on the ethanol-quenched hydrogels after 3 and 6 min of mineralization time. 2.6. Mesh Size Determination of the Hydrogels. Rheological measurements were performed with a dynamic mechanical analyzer in shear-mode (DMA-8000, PerkinElmer, USA) to measure the shear modulus of the hydrogels prior to mineralization. Samples with a diameter of 10 mm and a thickness of 3 mm were prepared for these measurements. Amplitude sweeps were performed as a function of the strain from 0.1 to 10% at a constant frequency of 0.5 Hz to determine the range of the linear response. Frequency sweeps (0.1−25 Hz) were conducted to measure the storage modulus G′ of each sample at a constant strain of 0.2%. The storage modulus G′ is related to the mesh size of the hydrogels, as described later in the main text.38 2.7. Agarose−Calcite Affinity. The interaction force between calcite and agarose was investigated in force measurements by atomic force microscopy (AFM) (JPK, Nanowizard, Germany). A cleaved calcite crystal was glued to a tipless cantilever (μMasch, HQ:CSC37, USA) with a stiffness of 156 mN/m using a micromanipulator (Sutter Instruments, MP-225, USA). The tip was approached at speeds ranging between 0.2 and 2 μm/s to the hydrogels. Sixty-four force− distance curves were collected over an area of 10 μm × 10 μm per speed on the hydrogels prepared with different agarose concentrations. 2.8. Simulation of Ion Diffusion through the Hydrogels during Crystal Growth. COMSOL Multiphysics (version 4.3b) was used to model the ion diffusion through a spherical sample of agarose hydrogel with a variable diameter. A spherical sink with a diameter of

The attenuation before mineralization (Aλ0) is given by the absorbance of the mineral-free hydrogel. Because A0λ was not observed to vary with the initial CaCl2 concentration, this value must remain constant upon mineralization: A0λ ≈ 0.01, 0.015, 0.0215, and 0.028 for 0.5, 1, 2, and 5 wt % hydrogels, respectively. In the spectrophotometric results, the difference in attenuation, i.e., ΔA= Aλ − A0λ, is shown as the time-dependent attenuation. During mineralization, the additional attenuation stems from the absorbance and the scattering of light by the precipitated solid in the hydrogel. At the selected wavelength, however, the absorbance of calcite is very small,34−36 and most of the attenuation stems from the scattering by the ACC nanoparticles. The number density of ACC nanoparticles is thus given by ΔAλ

c= 8π 3

(

2πnmed 4 λ

2

2

) ( mm +− 21 ) a6d 2

(4)

and the corresponding precipitated mass per unit volume of hydrogel is given by

m≈

cπa3 ρ 6 ACC

(5)

The validation of eq 5 via reference experiments with a known mass of scattering ACC particles showed the best agreement when using α2.73 instead of α3, which suggests that there is a deviation from Rayleigh scattering. This is not surprising because Rayleigh scattering is only strictly valid for objects that are smaller than 1/10 of the wavelength of the incident light,37 and the ACC particles in this work are larger (∼1/5 of the wavelength of the incident light). Therefore, it is more appropriate to use following modified empiric expression:

m≈

cπa2.73 ρ 6 ACC

(6)

To check the reproducibility between samples and the uniform mineralization within each hydrogel, five experiments were conducted per experimental condition. Two of them were time-resolved measurements of the attenuation in the center of two different hydrogels (averaged over an area with a diameter of ∼1 mm). Here, the attenuation was recorded every 5 s for a period of 6 h using OceanView software (Ocean Optics, v.1.5.0, USA). Three additional measurements were conducted at different positions within three additional hydrogel samples at selected points of time. In accompanying experiments, the fluid cell was fixed under a light microscope (Leica, MC170, Germany) to record time-resolved images of the mineralization process. The growth of four calcite crystals was recorded throughout the mineralization process in each of the duplicate experiments at two different magnifications (20× and 50×) and quantified by the average edge length; this number (four) was selected due to the low nucleation rate at the lowest concentrations. The crystal number density per unit area was measured at the end of the experiment in triplicate samples per experimental condition. Only the uppermost focal plane below the hydrogel’s surface was selected for the analysis of crystal size and number density. 2.3. Thermal Decomposition. The fraction of agarose embedded into the precipitated mineral was obtained by first isolating the crystals from the hydrogels, and then, by subjecting them to thermal decomposition in a thermogravimetric analyzer (TGA, PerkinElmer, Pyris 1, USA). The crystals were isolated by melting the hydrogels in boiling water and filtering the solids using a PVDF filter with a 0.2 μm mesh (Fisherbrand, USA). The solids were triplewashed with boiling water to remove any surface-adsorbed agarose, immersed in ethanol, and dried with a gentle stream of dry nitrogen. The isolated crystals (three samples from separate batches per condition) were then heated from 30 to 900 °C at a rate of 5 °C/min in the TGA. An average sample mass of 5.8 ± 3.7 mg was used for each TGA run. To rule out any changes of the crystals induced by the boiling water, the mineralized hydrogels were also incubated in a sodium hypochlorite solution (BICCA, 5% available chlorine) at 65 3194



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Figure 1. Representative spectrophotometric measurements. (a) Time-resolved attenuation during calcium carbonate mineralization in hydrogels with agarose concentrations of 0.5 wt % (gray triangles), 1 wt % (blue squares), 2 wt % (green diamonds), and 5 wt % (red circles) in 50 mM CaCO3 solutions and in 5 wt % agarose hydrogels at the concentrations of 5 and 15 mM CaCO3 solutions (black filled circles and crosses, respectively). (b) Mass of the precipitated CaCO3 per unit volume of hydrogel at 50 mM CaCO3. Dashed black line denotes the theoretical mass of CaCO3 per unit volume of hydrogel calculated according to stoichiometry. The average particle size α measured by DLS (Figure S3) was used for the calculation of the precipitated ACC mass according to eqs 1−5. Figures S2 and S4 in the SI show representative results for other experimental conditions. The cartoons represent the advancing front of ACC in the hydrogel, causing a characteristic turbidity in the sample starting on the hydrogel’s surface and advancing into the core, and a uniform crystal growth through dissolution of ACC and reprecipitation into calcite. 1 μm was placed in the center of the hydrogel volume, representing a calcite crystal. The attachment of the ionic specifies to the growing crystal was modeled by a first-order reaction J = kfC, where kf is the rate constant and C the concentration. The growth rate, G = J/ρc, ρc being the molar mass of calcite, was used to calculate the diameter of the crystal from Δd = GΔt, where Δt is the elapsed time of crystal growth. Two types of simulations were conducted: the simulation of the growth of calcite in the absence of ACC at an initial concentration of 5 mM, and the simulation of calcite in the presence of ACC, that is, while the concentration remains constant and equal to the solubility of ACC at the temperature of 25 °C.

3.1. Mineralization Pathway in the Agarose Hydrogels. Figure 1 shows representative measurements of the timedependent attenuation during mineralization in 0.5, 1, 2, and 5 wt % agarose hydrogels in a 50 mM CaCl2/Na2CO3 solution (abbreviated as 50 mM CaCO3), as well as in 5 wt % agarose hydrogels at the concentrations of 5 mM and 15 mM CaCO3; additional representative measurements are shown in Figure S2. Two different trends can be distinguished depending on the CaCO3 concentration. At the concentration of 5 mM, corresponding to a supersaturation with respect to calcite (σc) of 5.13 and with respect to ACC (σACC of 0.4 (Table S1)), the attenuation only increases modestly. Concurrently, light microscopy reveals that the growth of calcite crystals proceeds without the visible formation of any precursor phase. At higher CaCO3 concentrations (e.g., 50 mM in Figure 1), the attenuation remarkably increases during the initial 10−15 min of mineralization, which coincides with the hydrogel turning white and opaque. After achieving its maximum, the attenuation gradually decreases with time until a plateau is achieved at the end of the experiment, when the hydrogel recovers its transparency. The increase in attenuation and the notable turbidity of the hydrogels occur when a precursor phase precipitates during the first ∼15 min (see black dots in light microscopy images in Figure 2a), which transitions into calcite crystals while mineralization progresses. Raman microspectroscopy (Figures 2b−c and S5), FT-IR (Figure S6), and XRD (Figure S7) demonstrate the amorphous nature of this precursor phase, and the SEM images of the ethanol-quenched and critically dried hydrogels after 3 and 6 min of mineralization show the spherical morphology of the amorphous calcite carbonate nanoparticles (see an example in Figure 2f). The size distribution of the ACC nanoparticles is quite uniform (see the pronounced peaks at ∼90−130 nm in Figure S3), and it remains approximately unchanged between 3 and 6 min of mineralization, independently of agarose and CaCO3 concen-

3. RESULTS Agarose hydrogels were prepared at four different agarose concentrations (0.5, 1, 2, and 5% wt/wt, given as wt %) and each of them either with DI water (reference hydrogels, 0 mM) or with CaCl2 solution at concentrations of 5, 15, 50, 100, and 200 mM. A Na2CO3 solution with the same concentration as CaCl2 was then introduced into the fluid cell to induce mineralization, while spectrophotometric measurements and imaging of the hydrogels by optical light microscopy were conducted. Table S1 in the Supporting

( ),

Information (SI) shows the supersaturation σ = ln

IAP Ks

where IAP is the ion activity product and KS is the solubility product, with respect to calcite (σc) and ACC (σACC), calculated at the temperature of 25 °C using Visual MINTEQ Version 3.1. Under all conditions, mineralization occurs within the hydrogels, while the surrounding solution in the fluid cell remains free of visible crystals. The mass loss during the decomposition of calcite determined by TGA at the end of mineralization is used to determine the mineral content in the hydrogels, and it proves that the stoichiometric composition is achieved under all experimental conditions (Figure S1). A collection of the methods used in this study and of the main results is shown in Table S2. 3195



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Figure 2. (a) Time-resolved light microscopy images during mineralization of 5 wt % agarose hydrogels in 50 mM CaCl2/Na2CO3 (abbreviated as CaCO3 concentration). Scale bar = 50 μm. (b) 2D Raman microspectroscopy of an agarose hydrogel in 50 mM CaCO3 solution taken 3 min after the start of mineralization. The green area exhibits a Raman spectrum consistent with that of (c) ACC, while the red colored area exhibits the Raman spectrum of (d) calcite. Thus, ACC precipitates initially in the hydrogel and is visualized in light microscopy images by the black dots when they aggregate and are large enough. The calcite crystals are visible by light microscopy in (a) after 15 min. The arrows point at a clear depletion front of ACC. (e−g) SEM images of critically dried 1 wt % agarose hydrogels at different stages of mineralization in 50 mM CaCO3 solutions: (e) before mineralization, (f) after 3 min of mineralization, showing the precipitated ACC nanoparticles, and (g) at the end of the mineralization, showing various calcite crystals. Although the critically dried hydrogels were imaged by SEM before mineralization, the mesh size was not determined from these images due to the possible changes that the network undergoes upon critical drying.

As shown in Figure 1a, the attenuation ΔA, which accounts for the absorbance and the scattering of light by the precipitated mineral in the hydrogel, exhibits a pronounced initial increase with time followed by a decrease before the final plateau is achieved at equilibrium. However, the absorbance is expected to increase as the mass of precipitated calcium carbonate increases in the hydrogel, with a maximum value achieved at the end of the mineralization. This indicates that the scattering is the major contributor to the attenuation of light passing through the hydrogel, which is in agreement with the negligible absorptivity of calcium carbonate at the selected wavelength.40 Note that the final attenuation corresponds to the scattering caused by the calcite crystals, and therefore, the main contribution to the scattering is associated with ACC nanoparticles. The measured attenuation can thus be converted into the precipitated mass of ACC per unit volume of hydrogel using eqs 4−6 and the average particle size measured by DLS (Figure S3); it is assumed that the particle size remains constant, and only the number of particles changes during mineralization, first increasing until the peak is achieved and then decreasing. This estimation of the mineralization rate is shown in Figure 1b at the concentration of 50 mM CaCO3; see other representative measurements in Figure S4. The increase in mass with time during the first 5 min is used to evaluate the initial rate of ACC formation (see lines in Figure 1b), which is displayed in Figure 3a,b as a

tration. The precipitate observed by light microscopy is larger than these nanoparticles, which might result from the adventitious aggregation of some of the ACC nanoparticles in the hydrogel. While the attenuation decreases, microscopy images and Raman microspectroscopy display the growth of calcite crystals surrounded by regions depleted in ACC; eventually ACC is completely consumed, and only calcite crystals remain dispersed in the hydrogel. XRD confirms that calcite is the only crystalline polymorph that forms in the hydrogels over the investigated wide concentration range (Figure S8). 3.2. Influence of the Agarose Hydrogel on Mineralization. To rationalize the effect of the agarose concentration on the microstructure of the hydrogels, an oscillatory shear was applied to the hydrogels, and the storage modulus (G′) was measured. Increasing the agarose concentration from 0.5 wt % to 5 wt % thus leads to an increase of the storage modulus G′ from 2.2 to 97 kPa, which means that the strength of the network increases with agarose concentration. Scaling theory relates the storage modulus to the hydrogel’s mesh size (ξ) αkT according to G′ ≈ 3 , α being a constant, k the Boltzmann ξ

constant, and T the temperature.38,39 This relation predicts a reduction of the average mesh size by a factor of 3 with an increase in agarose concentration from 0.5 wt % to 5 wt %. 3196



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Figure 3. ACC formation rates as a function of (a) agarose concentration and (b) supersaturation of the solution with respect to ACC, σACC. The black markers in (a) (diamonds, squares, triangles, and crosses) are used to represent 15, 50, 100, and 200 mM CaCO3 concentrations, while the colored markers in (b) (gray triangles, blue squares, green diamonds, and red circles) represent the agarose concentrations 0.5, 1, 2, and 5 wt %, respectively.

function of agarose concentration and of supersaturation, respectively. Figure 3a illustrates that decreasing the agarose concentration (i.e., a greater mesh size) yields faster formation rates of ACC. In reference experiments in the absence of hydrogels (Figure S2), ACC was observed to form immediately, and the spectrophotometer was not able to record the attenuation associated with the formation process. This implies that the hydrogel network significantly slows down the formation of ACC, acting as a barrier for the diffusion of the carbonate ionsfrom the reservoir solution through the hydrogel, where it binds to calcium to form ACCand/or for the growth of the nanoparticles. As shown in Figure 3b, an increase in supersaturation promotes the formation rate of ACC; the data for 0.5 and 1 wt % hydrogels at the concentration of 200 mM CaCO3 are not shown because ACC forms so fast that it cannot be recorded by the spectrometer. 3.3. Calcite Precipitation. The number density of calcite crystals per unit area was determined at the end of the mineralization process by counting the number of crystals in light microscopy images (see representative images in Figure S9). Figure 4a shows the crystal density as a function of agarose concentration for each of the selected CaCO 3 concentrations. The crystal density passes through a minimum: at CaCO3 concentrations of at least 15 mM, the minimum in crystal density is observed at the agarose concentration of 2 wt %, while a less pronounced minimum in hydrogels with an agarose concentration of 1 wt % is observed at the CaCO3 concentration of 5 mM. The shift in the minimum coincides with the change in the mineralization pathway. First, the crystal density increases up to 2 orders of magnitude with CaCO3 concentration at concentrations of 15 mM and above. It also appears that the precipitation of calcite via the amorphous precursor promotes nucleation in hydrogels, except at agarose concentrations that are close to the minimum shown in Figure 4b, i.e., 1 wt % (blue squares) and 2 wt % (green diamonds). Further, the attenuation did not reveal the formation of an amorphous precursor phase that transforms into calcite at the CaCO3 concentration of 5 mM (note the lack of the characteristic increase and decrease in attenuation in Figure S2), and microscopy images did not show the black dots in Figure 2. The x-axis in Figure 4b has been selected to be an exponential function of the supersaturation with respect to

Figure 4. (a) Crystal density per unit area as a function of agarose concentration. Red arrows highlight the mechanisms of inhibition and promotion of calcite nucleation with increasing agarose concentration. Inhibition of crystal nucleation might result from the reduced likelihood of critical nuclei collisions and growth due to the presence of the agarose hydrogel network (so-called “shadow effect”). Empty markers in (a) are used to represent 15, 50, 100, and 200 mM CaCO3, and the filled circles represent the results for 5 mM CaCO3. (b) Crystal density as a function of exp(−1/σ2c ). Increasing values on the X-axis represent increasing concentration (5, 15, 50, 100, and 200 mM).

calcite, exp(−1/σc2), inspired by the relation between nucleation rate and supersaturation according to classical nucleation theory, i.e., Iv ≈ exp(−B/σ2c ).41 The deviation from 3197



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Figure 5. Crystal size of calcite as a function of time in (a) 0.5 wt % (black), (b) 1 wt % (blue), (c) 2 wt % (green), and (d) 5 wt % (red) agarose hydrogels at the CaCO3 concentrations of 5 (filled circles), 15 (diamonds), 50 (squares), 100 (triangles), and 200 mM (crosses). Empty symbols represent conditions where the amorphous precursor was detected prior to the precipitation of calcite, while the gray filled circles represent the conditions, at which ACC was not detected.

this relation is obvious when calcite forms via an amorphous precursor at the highest concentrations. Time-resolved light microscopy was used to measure the growth rate of single calcite crystals in the hydrogels. The crystal size as a function of time is shown in Figure 5a−d, each diagram displaying the results for a specific agarose concentration in the selected CaCO3 concentrations. The solid black lines represent a growth rate following a power law with an exponent between 0.25 and 0.35, while the insets show the initial 5 min of crystal growth, during which the growth rate is approximately constant and equal to ∼10.31 ± 0.71 nm/s at all conditions. Remarkably, the growth of the calcite single crystals with time does not depend significantly on CaCO3 concentrationdespite the 40-fold variation of concentrationand it is also similar in hydrogels of different composition (a−d); note that the error bars are larger than the difference across measurements. 3.4. Affinity between Calcite and Agarose Hydrogels. Representative images of isolated calcite crystals are shown in Figure 6a. Three distinct crystal morphologies were detected depending on both the agarose and the CaCO3 concentration. At the CaCO3 concentrations of 5 and 15 mM, the majority of calcite rhombohedra displays numerous nonequilibrium surface features such as highly terraced facets and distorted rhombohedral morphologies.42 At higher CaCO3 concentrations and low agarose concentrations, the crystalline rhombohedra display smoother facets. Increasing the agarose concentration to 2 and 5 wt % in 50 and 100 mM CaCO3

solutions and to 1 wt % in the case of 200 mM CaCO3 yields rhombohedra that evolve toward hopper-like structures with rough surfaces, pointed edges, and star-like morphologies. This increased surface roughness is interpreted to result from the interaction between the hydrogel matrix and the kink sites at specific facets of the growing crystals, which becomes more pronounced with increasing agarose concentration.43 The large (104) facets of the precipitated calcite crystals (Figure 6b) and the clean cleavage planes after light crushing (Figure 6c) support that the crystal growth of calcite proceeds through ACC dissolution and ion-by-ion attachment.42 The organic phase embedded within the crystals was quantified through TGA decomposition of the isolated crystals. The fraction of embedded agarose was estimated from the weight loss happening between 150 and 550 °C. A plateau was achieved at 550 °C, which extended to ∼600 °C, above which the decomposition of calcite started (Figure S1a). Figure 7a displays the weight percentage of agarose embedded in the calcite crystals as a function of the agarose concentration in the hydrogels. Importantly, the crystals precipitated in 5 mM CaCO3 solutions contain the largest amount of embedded agarose followed by those precipitated in 15 mM CaCO3 solutions. At higher CaCO3 concentrations (50−200 mM), the influence of the CaCO3 concentration on the amount of incorporated agarose is not statistically significant. However, the weight percentage of embedded agarose increases notably with agarose concentration between 0.5 and 2 wt %, while it 3198



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Figure 6. (a) SEM imaging of the calcite crystals precipitated in agarose hydrogels. Rows correspond to the different CaCO3 concentrations, while columns correspond to the selected agarose concentration. Green lines delineate conditions that yield small and highly roughened crystals, while red lines highlight conditions where hopper-crystal features develop. Scale bar = 50 μm. Calcite crystals were isolated by melting the agarose in boiling water, but similar crystal features were observed when a bleach treatment was used to remove the polymer (Figure S10). (b) Surface of calcite crystal precipitated in 1 wt % agarose in 100 mM CaCl2/Na2CO3. (c) Fracture faces of calcite crystals exhibit clean 104 facets after light crushing in a mortar.

significant attraction or repulsion. During retraction, the pull off force is negligible under all conditions. For comparison, Figure 7d shows analogous measurements between two calcite crystals, with a significant pull-off force (about 2 orders of magnitude larger), indicating the higher adhesion between the crystals in the same solution. The low adhesion energy between agarose and calcite suggests the action of a disjoining force between them, which is also expected to exist during the growth of calcite within the hydrogel network.

remains approximately constant at agarose concentrations higher than 2 wt %. To provide more insight into the incorporation mechanism of agarose into the crystal, the affinity of agarose to calcite was investigated by AFM indentation. A calcite crystal glued to an AFM tipless cantilever (Figure 7b) was approached to the hydrogel until it was in contact, and then, a maximum force of 20 nN was applied. While the applied force was increased, the calcite plate was increasingly pressed against the hydrogel and the indentation depth was measured. Upon reaching the maximum load, the cantilever was retracted to measure the adhesion between calcite and hydrogel. Figure 7c shows three representative force−indentation curves of a calcite crystal on a 1 wt % agarose hydrogel in equilibrium with a calcium carbonate saturated solution during extension (full markers, increasing load) and retraction (empty markers, decreasing load). The difference between the extension and retraction is due to the viscoelastic (delayed) deformation of the hydrogel. Before the calcite crystal makes contact with the hydrogel (see dashed arrow), the force is zero, meaning that there is no

4. DISCUSSION In this work, we have investigated CaCO3 mineralization in hydrogels with different agarose concentrations. Increasing the agarose concentration leads to a smaller mesh size and stronger agarose fibers,44 so that it modifies the hydrogel’s microstructure and its mechanical strength, while maintaining the chemical environment unmodified. The mineralization pathway is characterized by the formation of ACC prior to calcite in a concentration range from 15 to 200 mM CaCO3 (σACC ≈ 3199



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Figure 7. (a) Incorporated agarose within calcite crystals (by wt % fraction) as a function of the agarose concentration at the selected CaCO3 concentrations. Dashed lines correspond to calcite growth via the ACC precursor, while the solid line and filled markers represent the experimental conditions at which ACC was not observed. (b) Light microscopy images (top) of the calcite crystal glued to the AFM cantilever (red dashed square highlights the calcite crystal) and SEM image (bottom) of the glued calcite crystal. (c−d) Force−indentation curves upon extension (filled markers) and retraction (empty markers) at constant velocity of 20 nm/s measured with a calcite crystal glued to the AFM cantilever on (c) agarose hydrogels (1 wt %) and on (d) a calcite crystal fixed to the AFM stage, both in a saturated calcium carbonate solution.

2.1−5.3); only at the concentration of 5 mM CaCO3 (σACC ≈ 0.4) our experimental approach could not prove the precipitation of ACC. Several mechanisms can promote the stabilization of ACC: a high supersaturation has been shown to favor the formation of ACC, even in the absence of the hydrogel network. This was confirmed in our reference measurements, and it is in agreement with previous studies that have detected the formation of ACC in a similar concentration range (2−100 mM CaCO3) after mixing CaCl2 and Na2CO3 solutions,45 as in our experiments. In addition to this, hydrophilic polymers, such as agarose, have been reported to hinder the nucleation of calcite due to the high energetic barrier required to dehydrate the polymer, and thereby, to favor the stabilization of highly hydrated ACC.46 On the other hand, although agarose is a neutral polymer, divalent cations like calcium can bind to agarose.47,48 Analogous to studies on polyelectrolytes,49−51 an enhanced local supersaturation resulting from calcium binding to the organic network can also aid to stabilize ACC. The transformation of ACC to calcite may occur through either a dissolution−reprecipitation process or an aggregation of ACC nanoparticles as well as their attachment to crystals followed by solid-state transformation. In fact, calcite has been reported to grow through ACC attachment in certain biological hydrogel environments.42 It is to be noted that certain aggregation of ACC nanoparticles was shown in the light microscopy images (Figure 2a); however, the calcite crystals were not observed to preferentially grow within these regions, but instead they appeared randomly distributed.

Although light microscopy is not able to visualize either the nucleation of calcite or the aggregation of single ACC nanoparticles to the growing calcite crystals, it is reasonable to expect that the diffusion of the nanoparticles, and thereby their attachment to calcite, is hindered by the hydrogel network, since the mesh size is of the same order of magnitude or even smaller than the diameter of the ACC nanoparticles. The aggregation of ACC particles observed in light microscopy might happen in regions where the mesh size is larger than the average value (like in defects); however, this is a speculation at this point, and more work is needed to fully elucidate this phenomenon. Nevertheless, the SEM images of the calcite crystals (Figure 6b−c) do not exhibit the nanogranular imprint characteristic of such solid-state transformation.42,52 The cleavage planes of the crystals exhibit clean fracture faces instead of the conchoidal fracture patterns that typically occur in granular microstructures.42 Finally, the large (104) facets of the precipitated calcite crystals also support that calcite grows through ion-by-ion addition at the expense of ACC dissolution and not by solid-state transformation.42 Note that the calcium ions are initially uniformly distributed in the hydrogel, and then, the carbonate ions added to the reservoir diffuse into the hydrogel while ACC forms. The turbidity of the hydrogels caused by the ACC nanoparticles is observed to start close to the hydrogel’s surface and to advance to the center of the hydrogel, which indicates the presence of concentration gradients across the hydrogel. This is supported by the estimated kinetics. It is known that ion diffusion through a hydrogel depends on the hydrogel’s mesh size ξ.29 3200



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Figure 8. (a) Comparison between measured and simulated growth rate of calcite. The markers represent experimental results at the concentrations of 5 (filled circles) and 50 mM (empty squares) CaCO3 in 0.5 wt % hydrogels. The simulation of calcite growth was conducted by COMSOL Multiphysics. Here, an initial concentration of 4.12 mM was assumed, and it was considered to remain constant (dotted black line) to simulate the presence of ACC nanoparticles. The second simulation assumes that the concentration (initially 5 mM) decreases upon growth of calcite (gray line). The inset shows schematics of the spherical (hydrogel) volume and a close-up of the spherical sink with a simulated concentration profile surrounding it. (b) Concentration profile within the depletion zone and around the spherical sink representing the growing calcite crystal. The CaCO3 concentration, initially 5 mM, decreases uniformly over a period of 30 min in the absence of concentration gradients, until it achieves calcite’s solubility (0.13 mM).

first-order surface reaction growth with a rate constant, kf = 1 × 10−4 m/s, which lies well within the range of reported values for the growth rate constant of calcite.54,55 The fast (nonlimiting) diffusion of ions to the growing crystal through the hydrogel is reflected in the absence of concentration gradients, while the concentration drops uniformly with time (see the uniform colors at each point of time in Figure 8b). In the experiments at higher CaCO3 concentration (50 mM in Figure 8), where ACC is expected to dissolve in the regions surrounding the calcite crystals while calcite grows, the growth rate of calcite is initially similar to the simulated one, but it deviates at later times. The simulation shows that, when the concentration remains constant and equal to the solubility of ACC for extended periods of time, concentration gradients are absent as long as the diffusion length (the distance between calcite and ACC front) is small enough. A diffusion length larger than ∼200 μm (not shown), which would imply much lower nucleation rates than observed in agarose hydrogels, leads readily to the appearance of concentration gradients. The deviation of the experimental results from the dashed line can be caused by the decrease of concentration below the solubility of ACC after ∼15 min and also by the presence of concentration gradients. This also justifies that the growth rate becomes gradually more affected by the diffusivity of the hydrogel network, with a slower growth of calcite with a decrease in mesh size (Figure 5). Previous works concluded that the formation and dissolution of ACC is the rate-determining step for the growth of calcite during mineralization induced by equimolar solutions (50 mM) of CaCl2 and Na2CO3 in the presence of poly(acrylic acid) (PAA) and poly(styrenesulfonate-co-maleic acid) (PAAMA).56 The initial linear growth rate of calcite in our experiments (∼10 nm/s, insets in Figure 5) is in good agreement with the reported values for the growth rate of calcite in PAA and PAA-MA.56 This agreement is important, since the hydrogel-like environments in these works are chemically and structurally very different, and hence, discrepancies in the formation and dissolution kinetics of ACC and in the nucleation rate of calcite are very likely. Despite the different amounts of ACC nanoparticles feeding each single crystal in these multiple scenarios, the different

Figure 3a corroborates that the rate of formation of ACC in the selected hydrogels increases with mesh size (rf ≈ ξn, with n ranging from 0.19 to 0.27). Furthermore, it is also seen in Figure 3b that the rate of formation of ACC increases with supersaturation according to ∼σACC n , with an exponent n ranging from ∼3.4 (0.5−1 wt %) to ∼4 (2−5 wt %). These trends support that the formation of ACC is controlled by the diffusion of carbonate ions from the outside reservoir through the hydrogel, and it is, therefore, slowed down in hydrogels with smaller mesh size and supersaturation. The similarity of the growth rate independently of the initial supersaturation and of the mineralization pathway, i.e., through direct calcite precipitation or via the formation of an amorphous precursor, indicates that the growth of calcite at the expense of ACC dissolution is mainly dictated by the saturation concentration with respect to ACC (∼4.12 mM, σc = 4.8), which is close to the initial concentration during mineralization in 5 mM CaCO3 solutions. Furthermore, the growth rate of each single calcite crystal at the cost of ACC dissolution is remarkably similar across a wide spectrum of agarose concentrations (Figure 5), which suggests that ion diffusion through the polymer network is not the mechanism limiting the transformation of ACC into calcite. This is further supported by the numerical simulation described next. We hypothesize that for the growth of calcite not to be diffusion limited, it is necessary that the size of the ACCdepletion zones surrounding each single calcite crystal is small enough to enable a fast diffusion to the crystal from the surrounding ACC nanoparticles. Here, we assume that ACC nanoparticles are only present outside of the depletion zones (see arrows in Figure 2a). The size of these domains was observed to lie between 19 and 79 μm by light microscopy. To test this hypothesis, a commercial software (COMSOL)53 was used to model the growth of calcite in a spherical reservoir with diameters between 19 and 79 μm and diffusion coefficients ranging between 2 and 6 × 10−10 m2/s, corresponding to the diffusivity of the hydrogels with the selected range of agarose concentrations.11 For an initial concentration of 5 mM CaCO3 and a depletion diameter of 60 μm, Figure 8a illustrates that the growth rate observed in experiments can be reproduced in simulations considering a 3201



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in the elastic modulus G′ with an increase in agarose concentration reflects the increase in the strength of the polymer network.30,38,39 This, as well as the higher amount of the organic phase surrounding the crystal, can justify that the embedded agarose weight percentage increases with agarose concentration (Figure 7a). On the other hand, the higher inclusion of agarose in calcite crystals growing in 5 mM CaCO3 solutions compared to higher CaCO3 concentrations is intriguing, especially because the growth rates are so similar. The disjoining force between calcite and agarose could not be measured at concentrations higher than the solubility of calcite, because the supersaturated solution would cause the crystal to grow, which would disturb the force measurement. But the low adhesion between agarose and calcite (in a saturated solution) reveals the action of a disjoining force between polymer and crystal.12 From thermodynamics, it follows that a crystal surrounded by a supersaturated solution exerts a pressure against the confining surface (here the fiber), which is given by ∼RT/Vmσc, Vm being the molar volume, R the gas constant, and T the temperature.60,61 For a solution saturated with respect to ACC, the force of crystallization is very high (∼322 MPa). At the concentration of 5 mM CaCO3, the force of crystallization might be initially slightly greater (∼349 MPa), which could explain that the calcite crystals growing in this solution are initially more prone to incorporate agarose. Although this trend could reverse because the concentration decreases upon mineralization, we note that in 5 mM CaCO3, the crystal number density is up to 2 orders of magnitude smaller than at higher concentrations, and therefore, the decrease in concentration during crystallization might be delayed compared to higher concentrations, so that a high force of crystallization is maintained for longer periods of time. This is a speculation at this point that requires more dedicated measurements of the force of crystallization at different concentrations, the objective of our current work. The deviation of the crystal morphology from its equilibrium rhombohedral habit and the surface roughness and texturing at the lowest concentration (5 mM) also reflect the inclusion of the organic phase. It is possible that direct precipitation of calcite both from solution and via the amorphous precursor phase happen concurrently during mineralization at the concentration of 15 mM CaCO3, which would justify the intermediate amount of embedded agarose. Hence, the calcite crystals precipitated in 5−15 mM CaCO3 solutions feature the highest amounts of incorporated agarose per crystal and nonequilibrium habits, terracing, and surface roughness. In contrast, the precipitation of calcite through the amorphous precursor enables control of the polymer inclusions independently of the ionic strength over a wide range of conditions. There is, however, a limit to this control of mineralization via the amorphous phase in the hydrogels. While the growth rate and the agarose inclusions can still be maintained at the highest concentrations of CaCO3 used in this work, deviations from the equilibrium morphology, such as hopper-like crystals and star-like morphologies, are evident in the largest crystals in Figure 6a, which could point to diffusion-limited conditions during the later stages of the mineralization process.18,23 The chemical moieties and the spatial delineation provided by hydrogel-like environments have been reported to play an important role in biomineralization within living organisms. Plant cystoliths,42 for example, indefinitely stabilize ACC using a hydrogel-like environment to improve the scattering of light

hydrogel diffusivities and the varying diffusion lengths, the similarity in the growth rate of calcite implies that it cannot be limited by either the dissolution rate of ACC or the ion diffusion through the polymer networks, but instead it is controlled by surface reaction, like in our simulations. An important difference between calcium carbonate mineralization in the hydrogel and in the absence of the polymer network is the number of precipitated calcite crystals, which is orders of magnitude smaller in the latter (Figure S11). This clearly demonstrates that the hydrogel favors the nucleation of calcite. The nonmonotonic change of the crystal density with agarose concentration (Figure 4b) is intriguing and suggests the interplay of (at least) two competing mechanisms. It is possible that increasing the agarose concentration creates a “shadow” effect, where the agarose fibers hinder the random collisions between ion clusters preventing them from reaching a critical size, a necessary step for nucleation to occur.57 Concurrently, it is reasonable to expect that the number of low-surface energy nucleation sites (e.g., the amount of sulfate functional groups in agarose that can bind calcium ions more strongly) increases with agarose concentration, thereby, favoring calcite nucleation.58 It is also possible that the ACC nanoparticles are responsible for an additional “shadow” effect, and hence, for the more pronounced minimum in the crystal density observed at the higher CaCO3 concentrations compared to 5 mM and for the shift of the minimum from 1 wt % (in 5 mM CaCO3) to 2 wt %. Interestingly, similar mechanisms of promotion and inhibition of crystal formation have been previously reported in proteins and lead iodide crystallization in agarose hydrogels.58,59 As calcite grows under the conditions prescribed by ACC and the hydrogel environment, calcite incorporates agarose fibers. Figure 7a demonstrates that the incorporation of agarose into calcite strongly depends on the mineralization pathway, with a lower amount of embedded agarose occurring during mineralization at the highest CaCO3 concentrations, i.e., when the growth is mediated by ACC formation and dissolution. Here, the content of agarose in calcite is marginally larger (∼10%) than reported in previous studies of calcium carbonate mineralization in agarose hydrogels through the gas diffusion method in a similar CaCO3 concentration range (30−150 mM).12 In those previous works, however, it was not revealed if calcite formed through the formation of an amorphous precursor phase, and hence, this comparison should be made with caution. The inclusion of agarose during calcite growth has been rationalized to occur by two different mechanisms, a force and a mass balance, both at the crystal−organic interface.12 If the force of crystallization exerted by the crystal surpasses the disjoining pressure between crystal and fiber, and the fiber resists this force, the crystal grows around the fiber resulting in the inclusion of the polymer into the crystal. In contrast, weak fibers break and are pushed away and not incorporated into the crystal. On the other hand, the growth rate of the crystal has been proposed to dictate the mass balance. If the growth of the crystal is fast, then ions do not have enough time to diffuse to the crystal−agarose gap, starving the region of ions and causing the crystal to grow preferentially around the fiber. According to these two mechanisms, faster growth of calcite, higher force of crystallization, and/or stronger polymer network should lead to a higher amount of incorporated agarose into the crystals. In the investigated hydrogels of this work, the measured increase 3202



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within the leaf.62 In a distinct pathway, crustaceans63 mineralize disordered calcite crystallites within the hydrogel matrix to yield skeletal structures. Various implications for the biomineralization of tissues and skeletal components can be inferred from our work. The first one is that the hydrogel-like environment not only controls the mineralization pathway and kinetics via the modulation of ion diffusion, but also through the control of the nucleation rate of the crystalline phase, here calcite. Our results suggest that the nucleation rate is high enough to enable depletion zones of ACC surrounding each crystal to be small enough to hinder concentration gradients and diffusion-controlled growth so that crystals with a uniform size distribution precipitate throughout the hydrogel-like environment. The mineralization of CaCO3 via the amorphous precursor phase enables the amount of incorporated organic phase into the crystalline phase to be independent of variations in solution concentration, thereby exhibiting a high level of control of organic inclusion and morphology. The wide variety of chemical moieties available in synthetic hydrogels with a different affinity for the mineral phase, and the possibility of including soluble organic and biological matter in the hydrogel-like environment provides a wealth of opportunities to investigate diverse (bio)mineralization pathways.

a summary of the experimental methods, observables, and the main results in this work (PDF)

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Corresponding Author

*E-mail: [email protected]. Tel: +1 217 300 4380. ORCID

Rosa M. Espinosa-Marzal: 0000-0003-3442-2511 Funding

This work was supported by the National Science Foundation under Grant No. CMMI-1435920 and a TechnipFMC Fellowship, awarded to J.A.L.-B. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We would like to thank the Civil & Environmental Engineering Department at the University of Illinois Urbana−Champaign and its Research Experience for Undergraduates program for financial support for S.C., as well as Yijue Diao for her expertise and support making the AFM calcite tips.

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5. CONCLUSION Calcium carbonate mineralization was investigated in agarose hydrogels, and the effects of polymer network and solution supersaturation on mineralization were investigated. The hydrogel environment is responsible for the diffusion-limited formation of ACC within the hydrogel. The nucleation of calcite crystals is promoted by the hydrogel and through the formation of ACC, and the crystal density is sufficient to generate depletion zones smaller than the limiting value for a diffusion-controlled growth of calcite, according to simulations. In contrast, both the inclusion of the polymer into the calcite crystals, their morphology, and the rate of crystal growth remain unaffected by the supersaturation, demonstrating that the formation of the amorphous precursor phase affords control of CaCO3 mineralization in the hydrogel over a wide range of CaCO3 and agarose concentrations. Outside of this range, both the high levels of agarose incorporation at lower CaCO3 concentrations and the potential development of concentration gradients at high CaCO3 concentrations result in the expression of nonequilibrium morphologies of calcite. It is also proposed that agarose is playing multiple roles during mineralization, whether providing an organic scaffolding for the nucleation of calcite, a diffusion hindrance for ion transport, or facilitating chemical interactions between the polymer and ACC.

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AUTHOR INFORMATION

REFERENCES

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.cgd.9b00062. Calculated supersaturations by Visual MINTEQ, Raman spectroscopy, FT-IR, and XRD of hydrogels with ACC nanoparticles after 3 min of mineralization, XRD diffractograms, and SEM images of calcite crystals isolated from agarose hydrogels, light microscopy of crystal density, spectrophotometric measurements, DLS measurements, SEM imaging of hydrogels with ACC nanoparticles, and TGA measurements. A table contains 3203



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