Synergistic Biomineralization Phenomena Created by a Combinatorial Nacre Protein Model System

In the nacre or aragonite layer of the mollusk shell, proteomes that regulate both the early stages of nucleation and nano-to-mesoscale assembly of nacre tablets from mineral nanoparticle precursors exist. Several approaches have been developed to understand protein-associated mechanisms of nacre formation, yet we still lack insight into how protein ensembles or proteomes manage nucleation and crystal growth. To provide additional insights, we have created a proportionally defined combinatorial model consisting of two nacre-associated proteins, C-RING AP7 (shell nacre, Haliotis rufescens) and pseudo-EF hand PFMG1 (oyster pearl nacre, Pinctada fucata), whose individual in vitro mineralization functionalities are well-documented and distinct from one another. Using scanning electron microscopy, flow cell scanning transmission electron microscopy, atomic force microscopy, Ca(II) potentiometric titrations, and quartz crystal microbalance with dissipation monitoring quantitative analyses, we find that both nacre proteins are functionally active within the same mineralization environments and, at 1:1 molar ratios, synergistically create calcium carbonate mesoscale structures with ordered intracrystalline nanoporosities, extensively prolong nucleation times, and introduce an additional nucleation event. Further, these two proteins jointly create nanoscale protein aggregates or phases that under mineralization conditions further assemble into protein–mineral polymer-induced liquid precursor-like phases with enhanced ACC stabilization capabilities, and there is evidence of intermolecular interactions between AP7 and PFMG1 under these conditions. Thus, a combinatorial model system consisting of more than one defined biomineralization protein dramatically changes the outcome of the in vitro biomineralization process

Biomimetic intrafibrillar mineralization of type I collagen with intermediate precursors-loaded mesoporous carriers

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Nucleation in aqueous NaCl solutions shifts from 1-step to 2-step mechanism on crossing the spinodal

In this work, we use large-scale molecular dynamics simulations coupled to free energy calculations to identify for the first time a limit of stability (spinodal) and a change in the nucleation mechanism in aqueous NaCl solutions. This is a system of considerable atmospheric, geological and technical significance. We find that the supersaturated metastable NaCl solution reaches its limit of stability at sufficiently high salt concentrations, as indicated by the composition dependence of the salt chemical potential, indicating the transition to a phase separation by spinodal decomposition. However, the metastability limit of the NaCl solution does not correspond to spinodal decomposition with respect to crystallization. We find that beyond this spinodal, a liquid/amorphous separation occurs in the aqueous solution, whereby the ions first form disordered clusters. We term these clusters as "amorphous salt". We also identify a transition from one- to two-step crystallization mechanism driven by a spinodal. In particular, crystallization from aqueous NaCl solution beyond the spinodal is a two-step process, in which the ions first phase-separate into disordered amorphous salt clusters, followed by the crystallization of ions in the amorphous salt phase. In contrast, in the aqueous NaCl solution at concentrations lower than the spinodal, crystallization occurs via a one-step process, as the ions aggregate directly into crystalline nuclei. The change of mechanism with increasing supersaturation underscores the importance of an accurate determination of the driving force for phase separation. The study has broader implications on the mechanism for nucleation of crystals from solutions at high supersaturations

A classical view on nonclassical nucleation

Understanding and controlling nucleation is important for many crystallization applications. Calcium carbonate (CaCO_{3}) is often used as a model system to investigate nucleation mechanisms. Despite its great importance in geology, biology, and many industrial applications, CaCO_{3} nucleation is still a topic of intense discussion, with new pathways for its growth from ions in solution proposed in recent years. These new pathways include the so-called nonclassical nucleation mechanism via the assembly of thermodynamically stable prenucleation clusters, as well as the formation of a dense liquid precursor phase via liquid–liquid phase separation. Here, we present results from a combined experimental and computational investigation on the precipitation of CaCO_{3} in dilute aqueous solutions. We propose that a dense liquid phase (containing 4–7 H_{2}O per CaCO_{3} unit) forms in supersaturated solutions through the association of ions and ion pairs without significant participation of larger ion clusters. This liquid acts as the precursor for the formation of solid CaCO_{3} in the form of vaterite, which grows via a net transfer of ions from solution according to z Ca^{2+} + zCO_{3}^{2−} → z CaCO_{3}. The results show that all steps in this process can be explained according to classical concepts of crystal nucleation and growth, and that long-standing physical concepts of nucleation can describe multistep, multiphase growth mechanisms

Control of crystallization by polymer additives

Understanding how crystallization processes are controlled by polymer additives is relevant for a vastly diverse number of fields, for example in biomineralization, where the morphology of minerals is controlled by proteins or in scale inhibition strategies in industrial plants, in which macromolecules are added to prevent the precipitation of minerals. While numerous investigations have focused on elucidating additive-controlled crystallization, a detailed understanding of the underlying mechanisms is still desired. Herein, based on calcium carbonate as a mineral system, a comprehensive study on the manifold effects of selected polycarboxylate additives on the distinct species along the crystallization pathway is presented. Poly(glutamic acid) and poly(aspartic acid) are chosen as additives, as these polyaminoacids resemble biomineralization-associated peptides, as well as poly(acrylic acid), a commercially used scale inhibitor. Using potentiometric titrations, it is shown that even the basic interaction of polycarboxylates with calcium ions is more complex than commonly assumed. Quantitative determination of the Langmuir parameters of the binding process reveals that higher order effects and contributions arising from the whole polymer chain play a significant role, while the chemistry of the monomer unit constituting the polymer plays a subordinate role. The results put a question mark on whether the binding processes can be accurately described using solely the Langmuir binding model that assumes non-interacting binding sites. The investigation of the following stages of the crystallization pathway reveals that the polymer additives show the largest effect in the stabilization of liquid-like mineral precursors. A key step of this inhibition is the additive-driven binding of bicarbonates. Quantitative evaluation of ion association in the prenucleation regime reveals that more than 20% of bicarbonate species are bound in mineral precursors at pH 9.8, which can also be detected in isolated solid amorphous intermediates. Surprisingly, the protons introduced by bicarbonates are highly mobile, causing the formation of amorphous mineral ion conductors, which opens up possibilities for novel applications of mineral materials. The importance of liquid-like precursors for the mineral formation pathway is still highly debated in the community, and they are often ignored in the explanation of crystal formation. Using a refined gas diffusion method, it is demonstrated that liquid-like precursors show sufficient kinetic stability to be detected, both in presence of polymers and in additive-free systems. Observing the time dependent formation and transformation of the precursors shows that they play an important role in the early stages of crystallization and must be generally considered for the interpretation of gas diffusion experiments. Regarding the technological application of liquid-like minerals, a new and easily scalable synthesis method is presented, which solves existing limitations of the available synthesis methods. The “scalable controlled synthesis and utilization of liquid-like precursors for technological applications” (SCULPT) method effectively allows the isolation of the precursor on a gram scale and to access to the full potential of these mineral precursors for material synthesis. Implementing the gained insights into the current picture of nonclassical mineral formation, which was subject of many advancements in recent years, an updated view on additive-controlled mineralization is presented. The discoveries presented in this work are beneficial for the scientific and industrially-related communities far beyond the field of nucleation and crystallization mechanisms, such as materials chemistry, and improve the understanding of the mechanisms underlying biomineralization and mineral formation in general

“On demand” triggered crystallization of CaCO3 from solute precursor species stabilized by the water-in-oil microemulsion

Can we control the crystallization of solid CaCO3 from supersaturated aqueous solutions and thus mimic a natural process predicted to occur in living organisms that produce biominerals? Here we show how we achieved this by confining the reaction between Ca2+ and CO32- ions to the environment of nanosized water cores of water-in-oil microemulsions, in which the reaction between the ions is controlled by the intermicellar exchange processes. Using a combination of in situ small-angle X-ray scattering, high-energy X-ray diffraction, and low-dose liquid-cell scanning transmission electron microscopy, we elucidate how the presence of micellar interfaces leads to the formation of a solute CaCO3 phase/species that can be stabilized for extended periods of time inside micellar water nano-droplets. The nucleation and growth of any solid CaCO3 polymorph, including the amorphous phase, from such nano-droplets is prevented despite the fact that the water cores in the used microemulsion are highly supersaturated with respect to all known calcium carbonate solid phases. On the other hand the presence of the solute CaCO3 phase inside of the water cores decreases the rigidity of the micellar surfactant/water interface, which promotes the aggregation of micelles and the formation of large (>2 μm in diameter) globules. The actual precipitation and crystallization of solid CaCO3 could be triggered “on-demand” through the targeted removal of the organic-inorganic interface and hence the destabilization of globules carrying the CaCO3 solute

Characterization and tuning dollops in potable water

Calcium carbonate (CaCO₃) nucleation and scaling present challenges in industrial and environmental contexts, especially in potable water systems, where mineral scaling affects infrastructure and water quality. The traditional understanding of CaCO₃ nucleation has evolved to include non-classical pathways, including prenucleation clusters and dynamically ordered liquid-like oxyanion polymers (DOLLOPs). This research aimed to characterize DOLLOPs in potable water to mitigate scaling, but it unexpectedly shifted focus to intrinsic nanobubbles found in alkaline solutions.The research revealed that intrinsic CO₂ nanobubbles naturally form in alkaline aqueous solutions without external generation. Advanced techniques, such as Field-Flow Fractionation combined with Multi-Angle Light Scattering (FFF-MALS) and Zeta Nanoparticle Tracking Analysis (Z-NTA), characterized these nanobubbles in terms of size (approximately 100 nm in diameter), density, and zeta potential (negative charge). These nanobubbles are stable entities inherent to alkaline environments.Investigations into the influence of magnetic fields on nanobubble formation revealed that rotating magnetic fields significantly enhanced charged nanobubble formation, increasing their density and negative charge while impacting size distribution. This suggests that magnetic fields can control nanobubble populations in aqueous systems.The role of intrinsic nanobubbles in CaCO₃ formation was examined, revealing that charged nanobubbles inhibit nucleation by stabilizing the dense liquid calcium carbonate phase, preventing aggregation and delay in solid amorphous calcium carbonate (ACC) formation. Magnetically generated nanobubbles had a stronger effect, suggesting they influence the non-classical nucleation pathway by stabilizing intermediate states and delaying solid ACC formation.Additionally, Asymmetric Flow Field-Flow Fractionation coupled with Multi-Angle Light Scattering and Inductively Coupled Plasma Mass Spectrometry (AF4-MALS-ICP-MS) detected nanoparticles in drinking water samples, indicating potential DOLLOPs. Metals like magnesium were associated with small particles (1.5 to 10 nm), indicative of prenucleation clusters or DOLLOPs.The findings emphasize the crucial role of intrinsic nanobubbles in CaCO₃ nucleation and scaling in potable water systems. Recognizing nanobubbles as inherent components offers a fresh perspective on mineral scaling, providing novel approaches to manipulating CaCO₃ nucleation and scaling control in water treatment technologies

Characterization of Nanoparticles in Drinking Water Using Field-Flow Fractionation Coupled with Multi-Angle Light Scattering and Inductively Coupled Plasma Mass Spectrometry

The current absence of well-established and standardized methods for characterizing submicrometer- and nano-sized particles in water samples presents a significant analytical challenge. With the increasing utilization of nanomaterials, the potential for unintended exposure escalates. The widespread and persistent pollution of water by micro- and nanoplastics globally is a concern that demands attention, not only to reduce pollution but also to develop methods for analyzing these pollutants. Additionally, the analysis of naturally occurring nano entities such as bubbles and colloidal matter poses challenges due to the lack of systematic and consistent methodologies. This study presents Asymmetric Flow Field-Flow Fractionation (AF4) separation coupled with a UV-VIS spectrometer followed by Multi-Angle Light Scattering (MALS) for detection and size characterization of nanometric entities. It is coupled with an Inductively Coupled Plasma Mass Spectrometer (ICP-MS) for elemental analysis. Water samples from different sources, such as untreated mountain spring water, groundwater, and bottled drinking water, were analyzed. The system was calibrated using pure particle standards of different metallic compositions. Our study demonstrates the capability of AF4-UV-MALS-ICP-MS to detect metals such as Al, Ba, Cu, and Zn in particles of around 200 nm diameter and Mg associated with very small particles between 1.5 and 10 nm in different drinking water samples.</p

Quantum scale biomimicry of low dimensional growth: An unusual complex amorphous precursor route to TiO2 band confinement by shape adaptive biopolymer-like flexibility for energy applications

Crystallization via an amorphous pathway is often preferred by biologically driven processes enabling living species to better regulate activation energies to crystal formation that are intrinsically linked to shape and size of dynamically evolving morphologies. Templated ordering of 3-dimensional space around amorphous embedded non-equilibrium phases at heterogeneous polymer-metal interfaces signify important routes for the genesis of low-dimensional materials under stress-induced polymer confinement. We report the surface induced catalytic loss of P=O ligands to bond activated aromatization of C-C C=C and Ti=N resulting in confinement of porphyrin-TiO(2 )within polymer nanocages via particle attachment. Restricted growth nucleation of TiO2 to the quantum scale (˂= 2 nm) is synthetically assisted by nitrogen, phosphine and hydrocarbon polymer chemistry via self-assembly. Here, the amorphous arrest phase of TiO, is reminiscent of biogenic amorphous crystal growth patterns and polymer coordination has both a chemical and biomimetic significance arising from quantum scale confinement which is atomically challenging. The relative ease in adaptability of non-equilibrium phases renders host structures more shape compliant to congruent guests increasing the possibility of geometrical confinement. Here, we provide evidence for synthetic biomimicry akin to bio-polymerization mechanisms to steer disorder-to-order transitions via solvent plasticization-like behaviour. This challenges the rationale of quantum driven confinement processes by conventional processes. Further, we show the change in optoelectronic properties under quantum confinement is intrinsically related to size that affects their optical absorption band energy range in DSSC.This work was supported by the National Research Foundation of Korea (NRF) grant funded by Korea government (MEST) NRF-2012R1A1A2008196, NRF 2012R1A2A2A01047189, NRF 2017R1A2B4008801, 2016R1D1A1A02936936, (NRF-2018R1A4A1059976, NRF-2018R1A2A1A13078704) and NRF Basic Research Programme in Science and Engineering by the Ministry of Education (No. 2017R1D1A1B03036226) and by the INDO-KOREA JNC program of the National Research Foundation of Korea Grant No. 2017K1A3A1A68. We thank BMSI (A*STAR) and NSCC for support. SJF is funded by grant IAF25 PPH17/01/a0/009 funded by A* STAR/NRF/EDB. CSV is the founder of a spinoff biotech Sinopsee Therapeutics. The current work has no conflicting interests with the company. We would like to express our very great appreciation to Ms. Hyoseon Kim for her technical expertise during HRTEM imaging

Progress on the preparation of nanocrystalline apatites and surface characterization: Overview of fundamental and applied aspects

Nanocrystalline calcium phosphate apatites constitute the main inorganic part of hard tissues, and a growing focus is devoted to prepare synthetic analogs, so-called “biomimetic”, able to precisely mimic the morphological and physico-chemical features of biological apatite compounds. Both from fundamental and applied viewpoints, an accurate characterization of nanocrystalline apatites, including their peculiar surface features, and a deep knowledge of crystallization aspects are prerequisites to attempt understanding mineralization phenomena in vivo as well as for designing innovative bioactive materials that may then find applications in bone tissue engineering, either as self-supported scaffolds and fillers or in the form of coatings, but also in other domains such as drug delivery or else medical imaging. Also,interfacial phenomena are of prime importance for getting a better insight of biomineralization and for following the behavior of biomaterials in or close to their final conditions of use. In this view,both adsorption and ion exchange represent essential processes involving the surface of apatite nanocrystals, possibly doped with foreign elements or functionalized with organic molecules of interest. In this review paper, we will address these various points in details based on a large literature survey. We will also underline the fundamental physico-chemical and behavioral differences that exist between nanocrystalline apatites (whether of biological origin or their synthetic biomimetic analogs) and stoichiometric hydroxyapatite