Evaluation of correlated studies using liquid cell and cryo-transmission electron microscopy: Hydration of calcium sulphate and the phase transformation pathways of bassanite to gypsum.

Insight into the nucleation, growth and phase transformations of calcium sulphate could improve the performance of construction materials, reduce scaling in industrial processes and aid understanding of its formation in the natural environment. Recent studies have suggested that the calcium sulphate pseudo polymorph, gypsum (CaSO4 ·2H2 O) can form in aqueous solution via a bassanite (CaSO4 ·0.5H2 O) intermediate. Some in situ experimental work has also suggested that the transformation of bassanite to gypsum can occur through an oriented assembly mechanism. In this work, we have exploited liquid cell transmission electron microscopy (LCTEM) to study the transformation of bassanite to gypsum in an undersaturated aqueous solution of calcium sulphate. This was benchmarked against cryogenic TEM (cryo-TEM) studies to validate internally the data obtained from the two microscopy techniques. When coupled with Raman spectroscopy, the real-time data generated by LCTEM, and structural data obtained from cryo-TEM show that bassanite can transform to gypsum via more than one pathway, the predominant one being dissolution/reprecipitation. Comparisons between LCTEM and cryo-TEM also show that the transformation is slower within the confined region of the liquid cell as compared to a bulk solution. This work highlights the important role of a correlated microscopy approach for the study of dynamic processes such as crystallisation from solution if we are to extract true mechanistic understanding.

Enzymatically-controlled biomimetic synthesis of titania/protein hybrid thin films.

Although it is widely recognised that enzymes play a significant role in sculpting complex silica skeletons in marine sponges, the potential for exploiting enzymes in materials synthesis has not yet been fully harnessed. In this work we show that the digestive enzyme papain can self-assemble into monolayers on oxide surfaces, where they then drive the formation of crystalline titanium dioxide nanoparticles. This dual functionality of thin film formation and mineralization promotion has the potential to enable the construction of hierarchical inorganic/organic structures in the form of continuous amorphous titania/protein films which can be refined to 93% anatase post annealing. Additional control over the film thickness is afforded by layer-by-layer processing using a simple dip-coating approach. Papain's TiO2-mineralizing activity displays complex kinetics that deviates from the native Michaelis-Menten kinetic activity, yet deactivation studies demonstrate that this activity relies upon residues that are essential for catalytic site function. These parameters provide unique insight into enzymatic biomineralization, allowing a flexible route to achieving bioengineered titania heterostructures, and potentially providing a green-chemistry solution to photovoltaic cell development.

Virus-directed formation of electrocatalytically active nanoparticle-based Co3O4 tubes.

Spinel-type Co3O4 finds applications in a wide range of fields, including clean energy conversion, where nanostructured Co3O4 may provide a cost-efficient alternative to platinum- and iridium-based catalysts for electrocatalytic water-splitting. We here describe a novel strategy in which basic cobalt carbonate - a precursor to Co3O4 - is precipitated as sheet-like structures and microspheres covered with fine surface protrusions, via ammonium carbonate decomposition at room temperature. Importantly, these mild reaction conditions enable us to employ bio-inspired templating approaches to further control the mineral structure. Rod-like tobacco mosaic viruses (TMV) were used as biotemplates for mineral deposition, where we profit from the ability of Co(ii) ions to mediate the ordered assembly of the virus nanorods to create complex tubular superstructures of TMV/ basic cobalt carbonate. Calcination of these tubules is then achieved with retention of the gross morphology, and generates a hierarchically-structured solid comprising interconnected Co3O4 nanoparticles. Evaluation of these Co3O4 materials as electrocatalysts for the oxygen evolution reaction (OER) demonstrates that the activity of Co3O4 prepared by calcination of ammonia diffusion-grown precursors in both, the absence or presence of TMV exceeds that of a commercial nanopowder.

Crystal Nucleation without Supersaturation.

Classical nucleation theory (CNT) has been extensively employed to interpret crystal nucleation phenomena and postulates the formation of an ordered crystalline nucleus directly from vapor or solution. Here, we provide the first experimental demonstration of a two-step mechanism that facilitates deposition of crystals on solid surfaces from vapor. Crucially, this occurs from saturated vapor without the need for supersaturation, conditions that, according to CNT, cannot lead to direct deposition of crystals from vapor. Instead, the process relies on condensation of supercooled liquid in surface cavities below the melting point. Crystals then nucleate in this liquid, leading to rapid deposition of more solid. Such a mechanism has been postulated for atmospheric nucleation of ice on aerosol particles and may have analogies in the crystallization of biominerals via amorphous precursor phases.

Reconstitution of manganese oxide cores in horse spleen and recombinant ferritins.

The formation of Mn(III) oxyhydroxide (MnOOH) cores within the nanoscale cavity of the iron storage protein ferritin has been investigated by electron microscopy and visible absorption spectroscopy. At pH 8.9, discrete amorphous MnOOH cores were formed within horse spleen apoferritin at a range of metal:protein ratios, as well as in ferritin molecules seeded with a small ferrihydrite nucleus. Analysis of the resultant core size distributions showed that the reconstitution of horse spleen apoferritin with Mn(II) was similar to that observed previously for Fe(II) reconstitution in recombinant human L-chain ferritin, suggesting that horse spleen apoferritin does not exhibit Mn(II) oxidase activity at pH 8.9. Reconstitution with MnOOH shows essentially "all-or-nothing" behavior in which many protein molecules remain unmineralized whilst others are loaded to maximum capacity. Kinetic studies showed no significant differences between horse spleen ferritin, recombinant H- and L-chain homopolymers, and H-chain variants containing site-directed modifications at the ferroxidase and putative Fe nucleation centers. Our results indicate that the reconstitution of ferritin with MnOOH cores proceeds by a nonspecific pathway. We propose that the outer surface of the protein inhibits the development of MnOOH nuclei in bulk solution whereas the inner surface is inactive, enabling nucleation and growth to proceed unperturbed within the cavity. One possibility is that differences in the general polyelectrolyte properties of these two surfaces, rather than site-specific charges, account for the "Janus" behavior of the molecule. A similar mechanism might also increase the specificity of iron oxide mineralization in ferritins that lack ferroxidase centers.

Overproduction, purification and characterization of the Escherichia coli ferritin.

Recent studies have indicated that Escherichia coli possesses at least two iron-storage proteins, the haem-containing bacterioferritin and ferritin. The ferritin protein has been amplified 600-fold to 11-14% of total cell protein in a bfr mutant and purified to homogeneity with an overall yield of 13%. The cellular ferritin content remained relatively constant throughout the growth cycle and amplification was accompanied by a 2.5-fold increase in cellular iron content. The isolated ferritin contained 5-20 non-haem iron atoms/holomer and resembled the eukaryotic ferritins rather than the prokaryotic bacterioferritins in containing no haem. The 24 subunits of this ferritin (M(r) 19,400) assemble into a spherical protein shell (12 +/- 1 nm diameter, M(r) 465,000) which sequesters at least 2000 iron atoms in vitro to form an electron-dense iron core of 7.9 +/- 1 nm diameter. Electron-microscopic and Mössbauer spectroscopic studies with iron-loaded ferritin showed that the core can be either crystalline (ferrihydrite) or amorphous, depending on the absence or presence of phosphate, respectively. Mössbauer spectroscopy with intact E. coli revealed a novel-high spin Fe(II) component which is enhanced in bacteria amplified for ferritin but not in the parental strain. Western blotting showed that ferritin and bacterioferritin are immunologically distinct proteins. E. coli is thus an organism containing both a ferritin and a bacterioferritin and the relative roles of the two iron-storage proteins are discussed in this study.

Crystallization at Inorganic-organic Interfaces: Biominerals and Biomimetic Synthesis.

Crystallization is an important process in a wide range of scientific disciplines including chemistry, physics, biology, geology, and materials science. Recent investigations of biomineralization indicate that specific molecular interactions at inorganic-organic interfaces can result in the controlled nucleation and growth of inorganic crystals. Synthetic systems have highlighted the importance of electrostatic binding or association, geometric matching (epitaxis), and stereochemical correspondence in these recognition processes. Similarly, organic molecules in solution can influence the morphology of inorganic crystals if there is molecular complementarity at the crystal-additive interface. A biomimetic approach based on these principles could lead to the development of new strategies in the controlled synthesis of inorganic nanophases, the crystal engineering of bulk solids, and the assembly of organized composite and ceramic materials.

Magnetoferritin: in vitro synthesis of a novel magnetic protein.

The iron storage protein ferritin consists of a spherical polypeptide shell (apoferritin) surrounding a 6-nanometer inorganic core of the hydrated iron oxide ferrihydrite (5Fe2O3.9H2O). Previous studies have shown that the in vitro reconstitution of apoferritin yields mineral cores essentially identical to those of the native proteins. A magnetic mineral was synthesized within the nanodimensional cavity of horse spleen ferritin by the use of controlled reconstitution conditions. Transmission electron microscopy and electron diffraction analysis indicate that the entrapped mineral particles are discrete 6-nanometer spherical single crystals of the ferrimagnetic iron oxide magnetite (Fe3O4). The resulting magnetic protein, "magnetoferritin," could have uses in biomedical imaging, cell labeling, and separation procedures.