Functional mimicry of sea urchin biomineralization proteins with CaCO3-binding peptides selected by phage display.

The intricate process of biomineralization, e.g. in sea urchins, involves the precise interplay of highly regulated mineralization proteins and the spatiotemporal coordination achieved through compartmentalization. However, the investigation of biomineralization effector molecules, e.g. proteins, is challenging, due to their very low abundance. Therefore, we investigate the functional mimicry in the bioinspired precipitation of calcium carbonate (CaCO3) with artificial peptides selected from a peptide library by phage display based on peptide-binding to calcite and aragonite, respectively. The structure-directing effects of the identified peptides were compared to those of natural protein mixes isolated from skeletal (test) structures of two sea urchin species (Arbacia lixula and Paracentrotus lividus). The calcium carbonate samples deposited in the absence or presence of peptides were analyzed with a set of complementary techniques with regard to morphology, polymorph, and nanostructural motifs. Remarkably, some of the CaCO3-binding peptides induced morphological features in calcite that appeared similar to those obtained in the presence of the natural protein mixes. Many of the peptides identified as most effective in exerting a structure-directing effect on calcium carbonate crystallization were rich in basic amino acid residues. Hence, our in vitro mineralization study further highlights the important, but often neglected, role of positively charged soluble organic matrices associated with biological and bioinspired CaCO3 deposition.

Comparing the Colloidal Stabilities of Commercial and Biogenic Iron Oxide Nanoparticles That Have Potential In Vitro/In Vivo Applications.

For the potential in vitro/in vivo applications of magnetic iron oxide nanoparticles, their stability in different physiological fluids has to be ensured. This important prerequisite includes the preservation of the particles' stability during the envisaged application and, consequently, their invariance with respect to the transfer from storage conditions to cell culture media or even bodily fluids. Here, we investigate the colloidal stabilities of commercial nanoparticles with different coatings as a model system for biogenic iron oxide nanoparticles (magnetosomes) isolated from magnetotactic bacteria. We demonstrate that the stability can be evaluated and quantified by determining the intensity-weighted average of the particle sizes (Z-value) obtained from dynamic light scattering experiments as a simple quality criterion, which can also be used as an indicator for protein corona formation.

Size-controlled liquid phase synthesis of colloidally stable Co3O4 nanoparticles.

Spinel cobalt(ii,iii) oxide (Co3O4) represents a p-type semiconductor exhibiting promising functional properties in view of applications in a broad range of technological fields including magnetic materials and gas sensors as well as sustainable energy conversion systems based on photo- and electrocatalytic water splitting. Due to their high specific surface area, nanoparticle-based structures appear particularly promising for such applications. However, precise control over the diameter and the particle size distribution is required to achieve reproducible size-dependent properties. We herein introduce a synthetic strategy based on the decomposition of hydroxide precursors for the size-controlled preparation of purified Co3O4 nanoparticles with narrow size distributions adjustable in the range between 3-13 nm. The particles exhibit excellent colloidal stability. Their dispersibility in diverse organic solvents further facilitates processing (i.e. ligand exchange) and opens exciting perspectives for controlled self-assembly of the largely isometric primary particles into mesoscale structures. In view of potential applications, functional properties including absorption characteristics and electrocatalytic activity were probed by UV-Vis spectroscopy and cyclic voltammetry, respectively. In these experiments, low amounts of dispersed Co3O4 particles demonstrate strong light absorbance across the entire visible range and immobilized nanoparticles exhibit a comparably low overpotential towards the oxygen evolution reaction in electrocatalytic water splitting.

Long-Term Stability, Biocompatibility, and Magnetization of Suspensions of Isolated Bacterial Magnetosomes.

Magnetosomes are magnetic nanoparticles biosynthesized by magnetotactic bacteria. Due to a genetically strictly controlled biomineralization process, the ensuing magnetosomes have been envisioned as agents for biomedical and clinical applications. In the present work, different stability parameters of magnetosomes isolated from Magnetospirillum gryphiswaldense upon storage in suspension (HEPES buffer, 4 °C, nitrogen atmosphere) for one year in the absence of antibiotics are examined. The magnetic potency, measured by the saturation magnetization of the particle suspension, drops to one-third of its starting value within this year-about ten times slower than at ambient air and room temperature. The particle size distribution, the integrity of the surrounding magnetosome membrane, the colloidal stability, and the biocompatibility turn out to be not severely affected by long-term storage.

Mechanical adaptation of brachiopod shells via hydration-induced structural changes.

The function-optimized properties of biominerals arise from the hierarchical organization of primary building blocks. Alteration of properties in response to environmental stresses generally involves time-intensive processes of resorption and reprecipitation of mineral in the underlying organic scaffold. Here, we report that the load-bearing shells of the brachiopod Discinisca tenuis are an exception to this process. These shells can dynamically modulate their mechanical properties in response to a change in environment, switching from hard and stiff when dry to malleable when hydrated within minutes. Using ptychographic X-ray tomography, electron microscopy and spectroscopy, we describe their hierarchical structure and composition as a function of hydration to understand the structural motifs that generate this adaptability. Key is a complementary set of structural modifications, starting with the swelling of an organic matrix on the micron level via nanocrystal reorganization and ending in an intercalation process on the molecular level in response to hydration.

High-Yield Production, Characterization, and Functionalization of Recombinant Magnetosomes in the Synthetic Bacterium Rhodospirillum rubrum "magneticum".

Recently, the photosynthetic Rhodospirillum rubrum has been endowed with the ability of magnetosome biosynthesis by transfer and expression of biosynthetic gene clusters from the magnetotactic bacterium Magnetospirillum gryphiswaldense. However, the growth conditions for efficient magnetite biomineralization in the synthetic R. rubrum "magneticum", as well as the particles themselves (i.e., structure and composition), have so far not been fully characterized. In this study, different cultivation strategies, particularly the influence of temperature and light intensity, are systematically investigated to achieve optimal magnetosome biosynthesis. Reduced temperatures ≤16 °C and gradual increase in light intensities favor magnetite biomineralization at high rates, suggesting that magnetosome formation might utilize cellular processes, cofactors, and/or pathways that are linked to photosynthetic growth. Magnetosome yields of up to 13.6 mg magnetite per liter cell culture are obtained upon photoheterotrophic large-scale cultivation. Furthermore, it is shown that even more complex, i.e., oligomeric, catalytically active functional moieties like enzyme proteins can be efficiently expressed on the magnetosome surface, thereby enabling the in vivo functionalization by genetic engineering. In summary, it is demonstrated that the synthetic R. rubrum "magneticum" is a suitable host for high-yield magnetosome biosynthesis and the sustainable production of genetically engineered, bioconjugated magnetosomes.

Interface-mediated formation of basic cobalt carbonate/polyethyleneimine composite microscrolls by strain-induced self-rolling.

Polyethyleneimine aids the gas diffusion precipitation of nano-structured basic cobalt carbonate sheets at the air/solution interface. Upon drying, these mineral films undergo self-rolling into 3D coiled structures. Exploring this principle for the design of self-supported functional materials, porous Co3O4 spirals composed of interconnected nanoparticles are obtained by thermal conversion.

Towards standardized purification of bacterial magnetic nanoparticles for future in vivo applications.

Bacterial magnetosomes (MS) are well-defined membrane-enveloped single-domain iron oxide (magnetite) nanoparticles, which are susceptible to genetic and chemical engineering. Additionally, the possibility to manipulate these particles by external magnetic fields facilitates their application in biomedicine and biotechnology, e.g. as magnetic resonance imaging probes or for drug delivery purposes. However, current purification protocols are poorly characterized, thereby hampering standardized and reproducible magnetosome production and thus, reliable testing for in vivo applications. In that context, the establishment of reproducible particle isolation procedures as well as the identification of high quality control parameters and the evaluation of potential cytotoxic effects of purified particles are of major importance. In this study, we characterize a multi-step purification protocol for MS with regard to purity, iron content, size and polydispersity of magnetite particles. In addition, we address potential cytotoxic effects of isolated MS when incubated with mammalian cells. Overall, we provide a detailed overview of the process-structure relationship during the isolation of MS and thus, identify prerequisites for high-yield MS production and their future application in the biomedical and biotechnological field. STATEMENT OF SIGNIFICANCE: Magnetic nanoparticles are of increasing interest for a variety of biomedical and biotechnological applications. Due to their unprecedented material characteristics, bacterial magnetosomes represent a promising alternative to chemically synthesized iron oxide nanoparticles. As applications require well-defined, highly purified and fully characterized nanoparticles, reliable protocols are necessary for efficient and reproducible magnetosome isolation. In our study, we evaluate an improved magnetosome extraction procedure and monitor quality parameters such as particle size distribution, membrane integrity and purity of the suspension by a combination of physicochemical and biochemical methods. Furthermore, the cytotoxicity of the isolated magnetosomes is assessed using different cell lines. In summary, our study helps to establish prerequisites for many real-world applications of magnetosomes in the field of biotechnology and biomedicine.

An automated oxystat fermentation regime for microoxic cultivation of Magnetospirillum gryphiswaldense.

BACKGROUND: Magnetosomes produced by magnetotactic bacteria represent magnetic nanoparticles with unprecedented characteristics. However, their use in many biotechnological applications has so far been hampered by their challenging bioproduction at larger scales. RESULTS: Here, we developed an oxystat batch fermentation regime for microoxic cultivation of Magnetospirillum gryphiswaldense in a 3 L bioreactor. An automated cascade regulation enabled highly reproducible growth over a wide range of precisely controlled oxygen concentrations (1-95% of air saturation). In addition, consumption of lactate as the carbon source and nitrate as alternative electron acceptor were monitored during cultivation. While nitrate became growth limiting during anaerobic growth, lactate was the growth limiting factor during microoxic cultivation. Analysis of microoxic magnetosome biomineralization by cellular iron content, magnetic response, transmission electron microscopy and small-angle X-ray scattering revealed magnetosomal magnetite crystals were highly uniform in size and shape. CONCLUSION: The fermentation regime established in this study facilitates stable oxygen control during culturing of Magnetospirillum gryphiswaldense. Further scale-up seems feasible by combining the stable oxygen control with feeding strategies employed in previous studies. Results of this study will facilitate the highly reproducible laboratory-scale bioproduction of magnetosomes for a diverse range of future applications in the fields of biotechnology and biomedicine.

Probing the Nanostructure and Arrangement of Bacterial Magnetosomes by Small-Angle X-Ray Scattering.

Magnetosomes are membrane-enveloped single-domain ferromagnetic nanoparticles enabling the navigation of magnetotactic bacteria along magnetic field lines. Strict control over each step of biomineralization generates particles of high crystallinity, strong magnetization, and remarkable uniformity in size and shape, which is particularly interesting for many biomedical and biotechnological applications. However, to understand the physicochemical processes involved in magnetite biomineralization, close and precise monitoring of particle production is required. Commonly used techniques, such as transmission electron microscopy (TEM) or Fe measurements, allow only for semiquantitative assessment of the magnetosome formation without routinely revealing quantitative structural information. In this study, lab-based small-angle X-ray scattering (SAXS) is explored as a means to monitor the different stages of magnetosome biogenesis in the model organism Magnetospirillum gryphiswaldense SAXS is evaluated as a quantitative stand-alone technique to analyze the size, shape, and arrangement of magnetosomes in cells cultivated under different growth conditions. By applying a simple and robust fitting procedure based on spheres aligned in linear chains, it is demonstrated that the SAXS data sets contain information on both the diameter of the inorganic crystal and the protein-rich magnetosome membrane. The analyses corroborate a narrow particle size distribution with an overall magnetosome radius of 19 nm in Magnetospirillum gryphiswaldense Furthermore, the averaged distance between individual magnetosomes is determined, revealing a chain-like particle arrangement with a center-to-center distance of 53 nm. Overall, these data demonstrate that SAXS can be used as a novel stand-alone technique allowing for the at-line monitoring of magnetosome biosynthesis, thereby providing accurate information on the particle nanostructure.IMPORTANCE This study explores lab-based small-angle X-ray scattering (SAXS) as a novel quantitative stand-alone technique to monitor the size, shape, and arrangement of magnetosomes during different stages of particle biogenesis in the model organism Magnetospirillum gryphiswaldense The SAXS data sets contain volume-averaged, statistically accurate information on both the diameter of the inorganic nanocrystal and the enveloping protein-rich magnetosome membrane. As a robust and nondestructive in situ technique, SAXS can provide new insights into the physicochemical steps involved in the biosynthesis of magnetosome nanoparticles as well as their assembly into well-ordered chains. The proposed fit model can easily be adapted to account for different particle shapes and arrangements produced by other strains of magnetotactic bacteria, thus rendering SAXS a highly versatile method.

Virtually Wall-Less Tubular Sponges as Compartmentalized Reaction Containers.

Sponges are open cellular materials with numerous interesting features. However, the potential of compartmentalized sponges has not been explored although many new properties and applications could be envisioned. We found that compartmentalized fibrous ultraporous polymer sponges with superhydrophobic surfaces could be designed as virtually wall-less reaction containers. With this, for example, the efficient removal of CO2 from water and the controlled mineralization of calcium carbonate are possible. The high porosity (>99%) and superhydrophobicity make these sponges ideal candidates to hold alkanolamine solution for absorbing CO2 and exchange gas through the walls of the sponges. The tubular sponge exhibits a much higher evaporation rate than a glass tube with the same diameter due to the much larger contact area between water and air. Therefore, the spongy reaction container also possesses a much faster adsorption rate, smaller equilibration time and higher efficiency for CO2 adsorption than the glass tube container. In addition, these tubular sponges are also utilized to precipitate calcium carbonate by ammonium carbonate decomposition, which can control the deposition rates and products by tailoring the porosity and surface chemistry in the future. These new sponges provide an ideal basis for numerous new applications, for example, as breathable pipe lines for gas-liquid exchange, slag slurry carbonization, humidifier, and blood enricher.

Strain-relief by single dislocation loops in calcite crystals grown on self-assembled monolayers.

Most of our knowledge of dislocation-mediated stress relaxation during epitaxial crystal growth comes from the study of inorganic heterostructures. Here we use Bragg coherent diffraction imaging to investigate a contrasting system, the epitaxial growth of calcite (CaCO3) crystals on organic self-assembled monolayers, where these are widely used as a model for biomineralization processes. The calcite crystals are imaged to simultaneously visualize the crystal morphology and internal strain fields. Our data reveal that each crystal possesses a single dislocation loop that occupies a common position in every crystal. The loops exhibit entirely different geometries to misfit dislocations generated in conventional epitaxial thin films and are suggested to form in response to the stress field, arising from interfacial defects and the nanoscale roughness of the substrate. This work provides unique insight into how self-assembled monolayers control the growth of inorganic crystals and demonstrates important differences as compared with inorganic substrates.

Three-dimensional imaging of dislocation propagation during crystal growth and dissolution.

Atomic-level defects such as dislocations play key roles in determining the macroscopic properties of crystalline materials. Their effects range from increased chemical reactivity to enhanced mechanical properties. Dislocations have been widely studied using traditional techniques such as X-ray diffraction and optical imaging. Recent advances have enabled atomic force microscopy to study single dislocations in two dimensions, while transmission electron microscopy (TEM) can now visualize strain fields in three dimensions with near-atomic resolution. However, these techniques cannot offer three-dimensional imaging of the formation or movement of dislocations during dynamic processes. Here, we describe how Bragg coherent diffraction imaging (BCDI; refs 11, 12) can be used to visualize in three dimensions, the entire network of dislocations present within an individual calcite crystal during repeated growth and dissolution cycles. These investigations demonstrate the potential of BCDI for studying the mechanisms underlying the response of crystalline materials to external stimuli.

A critical analysis of calcium carbonate mesocrystals.

The term mesocrystal has been widely used to describe crystals that form by oriented assembly, and that exhibit nanoparticle substructures. Using calcite crystals co-precipitated with polymers as a suitable test case, this article looks critically at the concept of mesocrystals. Here we demonstrate that the data commonly used to assign mesocrystal structure may be frequently misinterpreted, and that these calcite/polymer crystals do not have nanoparticle substructures. Although morphologies suggest the presence of nanoparticles, these are only present on the crystal surface. High surface areas are only recorded for crystals freshly removed from solution and are again attributed to a thin shell of nanoparticles on a solid calcite core. Line broadening in powder X-ray diffraction spectra is due to lattice strain only, precluding the existence of a nanoparticle sub-structure. Finally, study of the formation mechanism provides no evidence for crystalline precursor particles. A re-evaluation of existing literature on some mesocrystals may therefore be required.

Confinement stabilises single crystal vaterite rods.

Single-crystals of vaterite, the least-stable anhydrous polymorph of CaCO3, are rare in biogenic and synthetic systems. We here describe the synthesis of high aspect ratio single crystal vaterite rods under additive-free conditions by precipitating CaCO3 within the cylindrical pores of track-etch membranes.

Bio-inspired formation of functional calcite/metal oxide nanoparticle composites.

Biominerals are invariably composite materials, where occlusion of organic macromolecules within single crystals can significantly modify their properties. In this article, we take inspiration from this biogenic strategy to generate composite crystals in which magnetite (Fe3O4) and zincite (ZnO) nanoparticles are embedded within a calcite single crystal host, thereby endowing it with new magnetic or optical properties. While growth of crystals in the presence of small molecules, macromolecules and particles can lead to their occlusion within the crystal host, this approach requires particles with specific surface chemistries. Overcoming this limitation, we here precipitate crystals within a nanoparticle-functionalised xyloglucan gel, where gels can also be incorporated within single crystals, according to their rigidity. This method is independent of the nanoparticle surface chemistry and as the gel maintains its overall structure when occluded within the crystal, the nanoparticles are maintained throughout the crystal, preventing, for example, their movement and accumulation at the crystal surface during crystal growth. This methodology is expected to be quite general, and could be used to endow a wide range of crystals with new functionalities.

Ion-association complexes unite classical and non-classical theories for the biomimetic nucleation of calcium phosphate.

Despite its importance in many industrial, geological and biological processes, the mechanism of crystallization from supersaturated solutions remains a matter of debate. Recent discoveries show that in many solution systems nanometre-sized structural units are already present before nucleation. Still little is known about the structure and role of these so-called pre-nucleation clusters. Here we present a combination of in situ investigations, which show that for the crystallization of calcium phosphate these nanometre-sized units are in fact calcium triphosphate complexes. Under conditions in which apatite forms from an amorphous calcium phosphate precursor, these complexes aggregate and take up an extra calcium ion to form amorphous calcium phosphate, which is a fractal of Ca(2)(HPO(4))(3)(2-) clusters. The calcium triphosphate complex also forms the basis of the crystal structure of octacalcium phosphate and apatite. Finally, we demonstrate how the existence of these complexes lowers the energy barrier to nucleation and unites classical and non-classical nucleation theories.