Direct Nanoscale Imaging Reveals the Mechanism by Which Organic Acids Dissolve Vivianite through Proton and Ligand Effects.

The coprecipitation of iron (Fe) and phosphorus (P) in natural environments limits their bioavailability. Plant root-secreted organic acids can dissolve Fe-P precipitates, but the molecular mechanism underlying mobilizing biogenic elements from highly insoluble inorganic minerals remains poorly understood. Here, we investigated vivianite (Fe3(PO4)2·8H2O) dissolution by organic acids (oxalic acid (OA), citric acid (CA), and 2'-dehydroxymugineic acid (DMA)) at three different pH values (4.0, 6.0, and 8.0). With increasing pH, the vivianite dissolution efficiency by OA and CA was decreased while that by DMA was increased, indicating various dissolution mechanisms of different organic acids. Under acidic conditions, weak ligand OA (HC2O4- > C2O42- at pH 4.0 and C2O42- at pH 6.0) dissolved vivianite through the H+ effect to form irregular pits, but under alkaline condition (pH 8.0), the completely deprotonated OA was insufficient to dissolve vivianite. At pH 4.0, CA (H2Cit- > HCit2- > H3Cit) dissolved vivianite to form irregular pits through a proton-promoted mechanism, while at pH 6.0 (HCit2- > Cit3-) and pH 8.0 (Cit3-), CA dissolved vivianite to form near-rhombohedral pits through a ligand-promoted mechanism. At three pH values ((H0)DMA3- > (H1)DMA2- at pH 4.0, (H0)DMA3- at pH 6.0, and (H0)DMA3- and one deprotonated imino at pH 8.0), strong ligand DMA dissolved vivianite to form near-rhombohedral pits via ligand-promoted mechanisms. Raman spectroscopy showed that the deprotonated carboxyl groups (COO-) and imino groups were bound to Fe on the vivianite (010) face. The surface free energy of vivianite coated with OA decreased from 29.32 mJ m-2 to 24.23 mJ m-2 and then to 13.47 mJ m-2 with increasing pH, and that coated with CA resulted in a similar pH-dependent vivianite surface free-energy decrease while that coated with DMA increased the vivianite surface free energy from 31.92 mJ m-2 to 39.26 mJ m-2 and then to 49.93 mJ m-2. Density functional theory (DFT)-based calculations confirmed these findings. Our findings provide insight into the mechanism by which organic acids dissolved vivianite through proton and ligand effects.

Oxide- and Silicate-Water Interfaces and Their Roles in Technology and the Environment.

Interfacial reactions drive all elemental cycling on Earth and play pivotal roles in human activities such as agriculture, water purification, energy production and storage, environmental contaminant remediation, and nuclear waste repository management. The onset of the 21st century marked the beginning of a more detailed understanding of mineral aqueous interfaces enabled by advances in techniques that use tunable high-flux focused ultrafast laser and X-ray sources to provide near-atomic measurement resolution, as well as by nanofabrication approaches that enable transmission electron microscopy in a liquid cell. This leap into atomic- and nanometer-scale measurements has uncovered scale-dependent phenomena whose reaction thermodynamics, kinetics, and pathways deviate from previous observations made on larger systems. A second key advance is new experimental evidence for what scientists hypothesized but could not test previously, namely, interfacial chemical reactions are frequently driven by "anomalies" or "non-idealities" such as defects, nanoconfinement, and other nontypical chemical structures. Third, progress in computational chemistry has yielded new insights that allow a move beyond simple schematics, leading to a molecular model of these complex interfaces. In combination with surface-sensitive measurements, we have gained knowledge of the interfacial structure and dynamics, including the underlying solid surface and the immediately adjacent water and aqueous ions, enabling a better definition of what constitutes the oxide- and silicate-water interfaces. This critical review discusses how science progresses from understanding ideal solid-water interfaces to more realistic systems, focusing on accomplishments in the last 20 years and identifying challenges and future opportunities for the community to address. We anticipate that the next 20 years will focus on understanding and predicting dynamic transient and reactive structures over greater spatial and temporal ranges as well as systems of greater structural and chemical complexity. Closer collaborations of theoretical and experimental experts across disciplines will continue to be critical to achieving this great aspiration.

Direct observation of humic acid-promoted hydrolysis of phytate through stabilizing a conserved catalytic domain in phytase.

As a potential phosphorus (P) pool, the enzymatic hydrolysis of organic phosphorus (Po) is of fundamental importance due to the release of bioavailable inorganic phosphate (Pi) for agronomic P sustainability. However, little is known about the role of soil organic matter (SOM) in the hydrolysis process of phytate by phytase and the subsequent chemical behaviors involving the hydrolysis product (Pi) at different soil interfaces. Here, by using liquid-cell atomic force microscopy (AFM), we present a model system to in situ quantify the nucleation kinetics of phytase-released Pi when precipitating with representative soil multivalent cations (Ca2+/Fe3+) on typical soil mineral/organic interfaces in the presence/absence of humic acid (HA), which involves complex phytase-interface-HA interactions. We observed that a higher HA concentration resulted in a faster nucleation rate of amorphous calcium/iron phosphate (ACP/AIP) on bare and organically-coated (-OH/-COOH) mica surfaces compared with the HA-free control. Besides, the nucleation rate of ACP/AIP induced by organic interfaces was much more significant than that induced by clay mineral interfaces. By combining enzyme activity/stability experiments and AFM-based PeakForce quantitative nanomechanical mapping (PF-QNM) measurements, we directly quantified the contribution of noncovalent phytase-HA interaction to the increase in enzymatic activity from complex phytase-interface-HA interactions. Furthermore, the direct complexation of phytase-HA resulted in the stabilization of a conserved active catalytic domain (ACD) in phytase through the enhanced formation of both an ordered, stereochemically-favored catalytic domain and an unordered non-catalytic domain, which was revealed by Raman secondary structure determination. The results provide direct insights into how HA regulates the catalytic activity of phytase controlling Po fates and how soil interfaces determine the behaviors of released Pi to affect its availability, and thereby contribute to P sustainability in soils.

Molecular mechanisms for the humic acid-enhanced formation of the ordered secondary structure of a conserved catalytic domain in phytase.

Changes in the secondary structure of phytase, particularly the conserved active catalytic domain (ACD, SRHGVRAPHD) are extremely important for the varied catalytic activity during hydrolyzing phytate in the presence of humic acid (HA). However, little is known about the molecular-scale mechanisms of how HA influences the secondary structure of ACD found in phytase. First, in situ surface-enhanced Raman spectroscopy (SERS) results show the secondary structure transformation of ACD from the unordered random coil to the ordered ß-sheet structure after treatment with HA. Then, we use an atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) technique that can in situ directly probe the single-molecule interaction of ACD with HA and underlying changes in ACD secondary structure in the approach-retraction cycles in real time. Based on the SMFS results, we further detect the HA-enhanced formation of H-bonding between amide groups in the ACD backbone after noncovalently interacting with HA in the absence of phytate. Following the addition of phytate, the calculated contour length (Lc) and the free energies (ΔGb) of functional groups within ACD(-1/2) binding to mica/HA collectively demonstrate the formation of the organized intermediate structural state of ACD following its covalent binding to phytate. These spectroscopic and single-molecule determinations provide the molecular-scale understanding regarding the detailed mechanisms of HA-enhancement of the ordered ß-sheet secondary structure of ACD through chemical functionalities in ACD noncovalently interacting with HA. Therefore, we suggest that similar studies of the interactions of other soil enzymes and plant nutrients may reveal predominant roles of dissolved organic matter (DOM) in controlling elemental cycling and fate for sustainable agriculture development.

Organically-bound silicon enhances resistance to enzymatic degradation and nanomechanical properties of rice plant cell walls.

Plant cell walls exhibit excellent mechanical properties, which form the structural basis for sustainable bioresources and multifunctional nanocelluloses. The wall nanomechanical properties of living cells through covalent modifications of hybrid inorganic elements, such as silicon, may confer significant influence on local mechano-response and enzymatic degradation. Here, we present a combination of ex situ measurements of enzyme-released oligosaccharide fragments using MALDI-TOF MS and in situ atomic force microscopy (AFM) imaging through PeakForce quantitative nanomechanical mapping of tip-functionalized single-molecule enzyme-polysaccharide substrate recognition and the nanoscale dissolution kinetics of individual cellulose microfibrils of living rice (Oryza sativa) cells following silicate cross-linking of cell wall xyloglucan. We find that xyloglucan-bound silicon enhances the resistance to degradation by cellulase and improves the wall nanomechanical properties in the elastic modulus at the single-cell level. The findings establish a direct link between an inorganic element of silicon and the nanoscale architecture of plant cell wall materials for sustainable utilization.

Dissolution and Precipitation Dynamics at Environmental Mineral Interfaces Imaged by In Situ Atomic Force Microscopy.

Chemical reactions at the mineral-solution interface control important interfacial processes, such as geochemical element cycling, nutrient recovery from eutrophicated waters, sequestration of toxic contaminants, and geological carbon storage by mineral carbonation. By time-resolved in situ imaging of nanoscale mineral interfacial reactions, it is possible to clarify the mechanisms governing mineral-fluid reactions.In this Account, we present a concise summary of this topic that addresses a current challenge at the frontier of understanding mineral interfaces and their importance to a wide range of mineral re-equilibration processes in the presence of a fluid aqueous phase. We have used real-time nanoscale imaging of liquid-cell atomic force microscopy (AFM) to observe the in situ coupling of the dissolution-precipitation process, whereby the dissolution of a parent mineral phase is coupled at mineral interfaces with the precipitation of another product phase, chemically different from the parent. These nanoscale observations allow for the identification of dissolution and growth rates through systematically investigating various minerals, including calcite (CaCO3), siderite (FeCO3), cerussite (PbCO3), magnesite (MgCO3), dolomite (CaMg(CO3)2), brushite (CaHPO4·2H2O), brucite (Mg(OH)2), portlandite (Ca(OH)2), and goethite (α-FeOOH), in various reacting aqueous fluids containing solution species, such as arsenic, phosphate, organo- or pyrophosphate, CO2, selenium, lead, cadmium, iron, chromium, and antimony. We detected the in situ replacement of these parent mineral phases by product phases, identified through a variety of analytical methods such as Raman spectroscopy, high-resolution transmission electron microscopy, and various X-ray techniques, as well as modeling by geochemical simulation using PHREEQC. As a consequence of the coupled processes, sequestration of toxic elements and hazardous species and inorganic and organic carbon, and limiting or promoting recovery of nutrients can be achieved at nano- and macroscopic scales.We also used in situ AFM to quantitatively measure the retreat rates of molecular steps and directly observe the morphology changes of dissolution etch pits on calcium phosphates in organic acid solutions present in most rhizosphere environments. By molecular modeling using density functional theory (DFT), we explain the origin of dissolution etch pit evolution through specific stereochemistry and molecular recognition and provide an energetic basis by calculating the binding energies of chemical functionalities on organic acids to direction-specific steps on mineral surfaces. In addition, we further quantified precipitation kinetics of calcium phosphates (Ca-P's) on typical mineral surfaces at the nanoscale in environmentally relevant solutions with various organic molecules, by measurements obtained from sequential images obtained by liquid-cell AFM. In situ dynamic force spectroscopy (DFS) was used to determine binding energies of single-molecules with different chemical functionalities found in natural organic matter at mineral-fluid interfaces. Quantifying molecular organo-mineral bonding or binding energies mechanistically explains phosphate precipitation and transformation. From DFS measurements, molecular-scale interactions of mineral-natural organic matter (DNA, proteins, and polysaccharides) associations were determined. With this powerful tool, single-molecule determinations of polysaccharide-amorphous iron oxide or hematite interactions provided the mechanistic origin of the phase- or facet-dependent adsorption. These systematic investigations and findings significantly contribute to a more quantitative prediction of the fate of nutrients and contaminants, chemical element cycling, and potential geological carbon capture and nuclear waste storage in aqueous environments.

Phosphorylated/Nonphosphorylated Motifs in Amelotin Turn Off/On the Acidic Amorphous Calcium Phosphate-to-Apatite Phase Transformation.

Amelotin (AMTN) as a matrix protein exerts a direct effect on biomineralization by modulating apatite (HAP) formation during the dental enamel maturation stage through the specific interaction of a potentially phosphorylated Ser-Ser-Glu-Glu-Leu (SSEEL) peptide fragment with calcium phosphate (Ca-P) surfaces. However, the roles of (non)phosphorylation of this evolutionarily conserved subdomain within AMTN remain poorly understood. Here, we show, by time-resolved atomic force microscopy (AFM) imaging of in situ HAP crystallization via the HPO42--rich amorphous calcium phosphate (acidic ACP), the on/off switching of the phase transformation process through a nonphosphorylation-to-phosphorylation transition of the SSEEL motif. Using high-resolution transmission electron microscopy (HRTEM), we observed that the acidic ACP phase is stabilized by the phosphorylated SSEEL motif, delaying its transformation to HAP, whereas the nonphosphorylated counterpart promotes HAP formation by accelerating the dissolution-recrystallization of the acidic ACP substrate. Dynamic force spectroscopy measurements demonstrate greater binding energies of nonphosphorylated SSEEL to the acidic ACP substrate by the formation of molecular peptide-ACP bonding, explaining the enhanced dissolution of the acidic ACP substrate by stronger complexion with surface Ca2+ ions. Our findings demonstrate direct evidence for the switching role of (non)phosphorylation of an evolutionarily conserved subdomain within AMTN in controlling the phase transition of growing enamel and designing tissue regeneration biomaterials.

Molecular Understanding of Humic Acid-Limited Phosphate Precipitation and Transformation.

Phosphorus (P) availability is widely assumed to be limited by the formation of metal (Ca, Fe, or Al) phosphate precipitates that are modulated by soil organic matter (SOM), but the SOM-precipitate interactions remain uncertain because of their environmental complexities. Here, we present a model system by quantifying the in situ nanoscale nucleation kinetics of calcium phosphates (Ca-Ps) on mica in environmentally relevant aqueous solutions by liquid-cell atomic force microscopy. We find that Ca-P precipitate formation is slower when humic acid (HA) concentration is higher. High-resolution transmission electron microscopy observations demonstrate that HA strongly stabilizes amorphous calcium phosphate (ACP), delaying its subsequent transformation to thermodynamically more stable phases. Consistent with the formation of molecular organo-mineral bonding, dynamic force spectroscopy measurements display larger binding energies of organic ligands with certain chemical functionalities on HA to the initially formed ACP than to mica that are responsible for stabilization of ACP through stronger HA-ACP interactions. Our results provide direct evidence for the proposed importance of SOM in inhibiting Ca-P precipitation/transformation. We suggest that similar studies of binding strength in SOM-Fe/Al-P may reveal how both organic matter and metal ions control P availability and fate, and thus the eventual P management for agronomical and environmental sustainability.

Molecular-Scale Investigations Reveal Noncovalent Bonding Underlying the Adsorption of Environmental DNA on Mica.

Mineral-soil organic matter (SOM including DNA, proteins, and polysaccharides) associations formed through various interactions, play a key role in regulating long-term SOM preservation. The mechanisms underlying DNA-mineral and DNA-protein/polysaccharide interactions at nanometer and molecular scales in environmentally relevant solutions remain uncertain. Here, we present a model mineral-SOM system consisting of mineral (mica)-nucleic acid (environmental DNA, eDNA)/protein (bovine serum albumin)/polysaccharide (alginate), and combine atomic force microscopy (AFM)-based dynamic force spectroscopy and PeakForce quantitative nanomechanical mapping using DNA-decorated tips. Single-molecule binding and adhesion force of eDNA to mineral and to mineral adsorbed by protein/polysaccharide reveal the noncovalent bonds and that systematically changing ion compositions, ionic strength, and pH result in significant differences in organic-organic and organic-mineral binding energies. Consistent with the bond-strength measurements, protein, rather than polysaccharide, promotes mineral-bound DNA molecules by ex situ AFM deposition observations in relatively high concentrations of divalent cation-containing acidic solutions. These molecular-scale determinations and nanoscale observations should substantially improve our understanding of how environmental factors influence the organic-mineral interfacial interactions through the synergy of collective noncovalent and/or covalent bonds in mineral-organic associations.

Direct Observations of the Occlusion of Soil Organic Matter within Calcite.

Global soil carbon cycling plays a key role in regulating and stabilizing the earth's climate change because of soils with amounts of carbon at least three times greater than those of other ecological systems. Soil minerals have also been shown to underlie the persistence of soil organic matter (SOM) through both adsorption and occlusion, but the microscopic mechanisms that control the latter process are poorly understood. Here, using time-resolved in situ atomic force microscopy (AFM) to observe how calcite, a representative mineral in alkaline soils, interacts with humic substances, we show that following adsorption, humic substances are gradually occluded by the advancing steps of spirals on the calcite (1014) face grown in relatively high supersaturated solutions, through the embedment, compression, and closure of humic substance particles into cavities. This occlusion progress is inhibited by phytate at high concentrations (10-100 µM) due to the formation of phytate-Ca precipitates on step edges to prevent the step advancement, whereas phytate at relatively low concentrations (≤1 µM) and oxalate at high concentrations (100 µM) have little effect on this process. These in situ observations may provide new insights into the organo-mineral interaction, resulting in the incorporation of humic substances into minerals with a longer storage time to delay degradation in soils. This will improve our understanding of carbon cycling and immobilization in soil ecological systems.

Underlying Role of Brushite in Pathological Mineralization of Hydroxyapatite.

The majority of human kidney stones are composed of multiple calcium oxalate crystals with variable amounts of brushite [dicalcium phosphate dihydrate (DCPD)] and hydroxyapatite (HAP) as a nucleus, in which fluid-mediated dissolution and reprecipitation may result in the phase transformation of DCPD to HAP. However, the underlying mechanisms of the phase transition and its modulation by natural inhibitors, such as osteopontin (OPN) proteins, remain poorly understood. Here, the in vitro formation of new phases on the DCPD (010) surface is observed in situ using atomic force microscopy in a simulated hypercalciuria milieu. We demonstrate the presence of an acidic amorphous calcium phosphate (ACP) phase with a characteristic Raman band of ν1HPO42- and the octacalcium phosphate (OCP)-like phase during the transformation process. High-resolution transmission electron microscopy analyses also confirm the existence of OCP and HAP within an amorphous matrix phase. In support of clinical observations, we further demonstrate the inhibitory effect of OPN peptide segments on the dissolution of DCPD and reprecipitation of acidic ACP. The definition of respective roles of DCPD and OPN thereby provides insights into the control of nucleus formation and subsequent inhibition of pathological mineralization.

Inhibition of Spiral Growth and Dissolution at the Brushite (010) Interface by Chondroitin 4-Sulfate.

Modulation of mineralization and demineralization of calcium phosphates (Ca-Ps) with organic macromolecules is a critical process which prevents human kidney stone disease. As a long unbranched polysaccharide of urinary glycosaminoglycans, chondroitin 4-sulfate (Ch4S) has been shown to play an essential role in inhibiting the formation of kidney stones. However, the mechanism of the role of Ch4S remains poorly understood. Here, we used in situ atomic force microscopy to observe the growth and dissolution of spirals on brushite (CaHPO4·2H2O) (010) surfaces. The results show that Ch4S preferentially inhibits the [101]Cc step growth/dissolution by step pinning. This step-specific effect appears to be related to specific binding of Ch4S to Ca sites, as the observed inhibition is not seen in other crystallographic directions where there are fewer Ca terminations. Moreover, Ch4S promotes an increase in the terrace width of [101̅]Cc by the modification of the interfacial energies of the step edge. These in vitro direct observations of Ch4S modulating brushite mineralization and demineralization reveal a dual control of both step kinetics and interfacial energy.

Humic Acids Limit the Precipitation of Cadmium and Arsenate at the Brushite-Fluid Interface.

Bioavailability and mobility of cadmium (Cd2+) and arsenate (As5+) in soils can be effectively lowered through the dissolution of brushite (dicalcium phosphate dihydrate, CaHPO4·2H2O) coupled with the precipitation of a more stable mineral phase containing both Cd and As. Due to the ubiquitous presence of humic acid (HA) in soil environments, it is more complex to predict the fate of dissolved Cd and As during such sequestration. Here, we used in situ atomic force microscopy (AFM) to image the kinetics of simultaneous precipitation of Cd and As at the brushite-fluid interface in the presence of HA. Results show that HA inhibits the formation of both amorphous and crystalline Cd(5- x)Ca x(PO4)(3- y)(AsO4) y(OH) on the (010) face of brushite. A combination of X-ray photoelectron spectroscopy (XPS) and real-time surface-enhanced Raman spectroscopy (SERS) reveals that part of As5+ reduction into As3+ with HA and [HA-Cd] complexation occurs, modulating the concentrations of free Cd2+ and As5+ ions to inhibit subsequent precipitation of a Cd(5- x)Ca x(PO4)(3- y)(AsO4) y(OH) phase on the dissolving brushite surface. A combination of AFM imaging, SERS analyses, and PhreeqC simulations suggests that environmentally relevant humic substances can limit the precipitation of Cd and As at mineral surfaces through a mechanism of oxidation/reduction and aqueous/surface complexation. This may exacerbate the transportation of these contaminants into waters by subsurface fluid flow, and research attempts to weaken the negative effect of HA are needed.

Dynamics and Molecular Mechanism of Phosphate Binding to a Biomimetic Hexapeptide.

Phosphorus (P) recovery from wastewater is essential for sustainable P management. A biomimetic hexapeptide (SGAGKT) has been demonstrated to bind inorganic P in P-rich environments, however the dynamics and molecular mechanisms of P-binding to the hexapeptide still remain largely unknown. We used dynamic force spectroscopy (DFS) to directly distinguish the P-unbound and P-bound SGAGKT adsorbed to a mica (001) surface by measuring the single-molecule binding free energy (Δ Gb). Using atomic force microscopy (AFM) to determine real-time step retreat velocities of triangular etch pits formed at the nanoscale on the dissolving (010) face of brushite (CaHPO4·2H2O) in the presence of SGAGKT, we observed that SGAGKT peptides promoted in situ dissolution through an enhanced P-binding driven by hydrogen bonds in a P-loop being capable of discriminating phosphate over arsenate, concomitantly forming a thermodynamically favored SGAGKT-HPO42- complexation at pH 8.0 and relatively low ionic strength, consistent with the DFS and isothermal titration calorimetry (ITC) determinations. The findings reveal the thermodynamic and kinetic basis for binding of phosphate to SGAGKT and provide direct evidence for phosphate discrimination in phosphate/arsenate-rich environments.

Direct Observation of Simultaneous Immobilization of Cadmium and Arsenate at the Brushite-Fluid Interface.

Cadmium (Cd2+) and Arsenate (As5+) are the main toxic elements in soil environments and are easily taken up by plants. Unraveling the kinetics of the adsorption and subsequent precipitation/immobilization on mineral surfaces is of considerable importance for predicting the fate of these dissolved species in soils. Here we used in situ atomic force microscopy (AFM) to image the dissolution on the (010) face of brushite (dicalcium phosphate dihydrate, CaHPO4·2H2O) in CdCl2- or Na2HAsO4-bearing solutions over a broad pH and concentration range. During the initial dissolution processes, we observed that Cd or As adsorbed on step edges to modify the morphology of etch pits from the normal triangular shape to a four-sided trapezium. Following extended reaction times, the respective precipitates were formed on brushite through a coupled dissolution-precipitation mechanism. In the presence of both CdCl2 and Na2HAsO4 in reaction solutions at pH 8.0, high-resolution transmission electron microscopy (HRTEM) showed a coexistence of both amorphous and crystalline phases, i.e., a mixed precipitate of amorphous and crystalline Cd(5- x)Ca x(AsO4)(3- y)(PO4) yOH phases was detected. These direct dynamic observations of the transformation of adsorbed species to surface precipitates may improve the mechanistic understanding of the calcium phosphate mineral interface-induced simultaneous immobilization of both Cd and As and subsequent sequestration in diverse soils.

Peridotite weathering is the missing ingredient of Earth's continental crust composition.

The chemical composition of the continental crust cannot be adequately explained by current models for its formation, because it is too rich in Ni and Cr compared to that which can be generated by any of the proposed mechanisms. Estimates of the crust composition are derived from average sediment, while crustal growth is ascribed to amalgamation of differentiated magmatic rocks at continental margins. Here we show that chemical weathering of Ni- and Cr-rich, undifferentiated ultramafic rock equivalent to ~1.3 wt% of today's continental crust compensates for low Ni and Cr in formation models of the continental crust. Ultramafic rock weathering produces a residual that is enriched in Ni and also silica. In the light of potentially large volumes of ultramafic rock and high atmospheric CO2 concentrations during the Archean, chemical weathering must therefore have played a major role in forming compositionally evolved components of the early Earth's crust.

Mechanisms of Modulation of Calcium Phosphate Pathological Mineralization by Mobile and Immobile Small-Molecule Inhibitors.

Potential pathways for inhibiting crystal growth are via either disrupting local microenvironments surrounding crystal-solution interfaces or physically blocking solute molecule attachment. However, the actual mode of inhibition may be more complicated due to the characteristic time scale for the inhibitor adsorption and relaxation to a well-bound state at crystal surfaces. Here we demonstrate the role of citrate (CA) and hydroxycitrate (HCA) in brushite (DCPD, CaHPO4·2H2O) crystallization over a broad range of both inhibitor concentrations and supersaturations by in situ atomic force microscopy (AFM). We observed that both inhibitors exhibit two distinct actions: control of surface crystallization by the decrease of step density at high supersaturations and the decrease of the [1̅00]Cc step velocity at high inhibitor concentration and low supersaturation. The switching of the two distinct modes depends on the terrace lifetime, and the slow kinetics along the [1̅00]Cc step direction provides specific sites for the newly formed dislocations. Molecular modeling shows the strong HCA-crystal interaction by molecular recognition, explaining the AFM observations for the formation of new steps and surface dissolution along the [101]Cc direction due to the introduction of strong localized strain in the crystal lattice. These direct observations highlight the importance of the inhibitor coverage on mineral surfaces, as well as the solution supersaturation in predicting the inhibition efficacy, and reveal an improved understanding of inhibition of calcium phosphate biomineralization, with clinical implications for the full therapeutic potential of small-molecule inhibitors for kidney stone disease.

Sequestration of Antimony on Calcite Observed by Time-Resolved Nanoscale Imaging.

Antimony, which has damaging effects on the human body and the ecosystem, can be released into soils, ground-, and surface waters either from ore minerals that weather in near surface environments, or due to anthropogenic releases from waste rich in antimony, a component used in batteries, electronics, ammunitions, plastics, and many other industrial applications. Here, we show that dissolved Sb can interact with calcite, a widespread carbonate mineral, through a coupled dissolution-precipitation mechanism. The process is imaged in situ, at room temperature, at the nanometer scale by using an atomic force microscope equipped with a flow-through cell. Time-resolved imaging allowed following the coupled process of calcite dissolution, nucleation of precipitates at the calcite surface and growth of these precipitates. Sb(V) forms a precipitate, whereas Sb(III) needs to be oxidized to Sb(V) before being incorporated in the new phase. Scanning-electron microscopy and Raman spectroscopy allowed identification of the precipitates as two different calcium-antimony phases (Ca2Sb2O7). This coupled dissolution-precipitation process that occurs in a boundary layer at the calcite surface can sequester Sb as a solid phase on calcite, which has environmental implications as it may reduce the mobility of this hazardous compound in soils and groundwaters.

Energetic Basis for Inhibition of Calcium Phosphate Biomineralization by Osteopontin.

Calcium oxalate kidney stones form attached to Randall's plaques (RP), calcium phosphate (Ca-P) deposits on the renal papillary surface. Osteopontin (OPN) suppresses crystal growth in the complex process of urinary stone formation, but the inhibitory role of active domains of OPN involved in the initial formation of the RPs attached to epithelial cells has yet to be clarified. Here we demonstrate the thermodynamic basis for how OPN sequences regulate the onset of Ca-P mineral formation on lipid rafts as a model membrane. We first quantify the kinetics of hydroxyapatite (HAP) nucleation on membrane substrates having liquid-condensed (LC) and liquid-expanded (LE) phases using in situ atomic force microscopy (AFM). We find that rates are sequence-dependent, and the thermodynamic barrier to nucleation is reduced by minimizing the interfacial free energy γ. Combined with single-molecule determination of the binding energy (ΔGB) of the OPN peptide segments adsorbed to the HAP (100) face, we show a linear relationship of γ and ΔGB, suggesting that the increase in the nucleation barriers correlates with strong peptide-crystal nuclei binding. These findings reveal fundamental energetic clues for inhibition of membrane-mediated nucleation by sequence motifs and subdomains within the OPN protein through spatial location of charged moieties and provide insight connecting peripheral cell membranes to pathological mineralization.