Bioinspired synthesis and characterization of organic/inorganic nanocomposite materials

In Nature, hybrid materials with hierarchical structure are formed by biomineralization of organic macromolecules that act as templates for the nucleation and/or growth of the inorganic component. The building blocks of the natural organic macromolecules provide the template architectures that result in chemical and morphological diversity in the inorganic phases. Inspired by the formation of biominerals in living organisms, novel organic-inorganic hybrid materials have been designed and developed by biomimetic routes. There is a growing interest in using synthetic polymers, engineered proteins, and various polymer-based hybrid architectures as templates for bioinspired synthesis. In this work, we have used amphiphilic block copolymers as well as block copolymer-protein conjugates that undergo hierarchical self-assembly to form nanoscale micelles and macroscale gels as templates for controlled nanocomposite formation within the polymeric matrix. The amphiphilic block copolymers that were generated based on Pluronics are unique systems that can reversibly self-assemble into macroscale elastic solids in solution, based on pH and temperature changes. The efforts were focused on three systems--calcium phosphate nanocomposites, zirconia nanocomposites, and magnetic nanocomposites. We have developed a robust method with control over the formation as well as placement of an inorganic phase in the nanocomposite structure, for a variety of different inorganic nanoparticles, such as calcium phosphate, zirconia and magnetic nanoparticles. The future work will be focused on using biominerlization proteins to create functional dynamic magnetic materials and nanostructures both in solution and on surfaces

Nucleation of Iron Oxide Nanoparticles Mediated by Mms6 Protein in Situ

Biomineralization proteins are widely used as templating agents in biomimetic synthesis of a variety of organic-inorganic nanostructures. However, the role of the protein in controlling the nucleation and growth of biomimetic particles is not well understood, because the mechanism of the bioinspired reaction is often deduced from ex situ analysis of the resultant nanoscale mineral phase. Here we report the direct visualization of biomimetic iron oxide nanoparticle nucleation mediated by an acidic bacterial recombinant protein, Mms6, during an in situ reaction induced by the controlled addition of sodium hydroxide to solution-phase Mms6 protein micelles incubated with ferric chloride. Using in situ liquid cell scanning transmission electron microscopy we observe the liquid iron prenucleation phase and nascent amorphous nanoparticles forming preferentially on the surface of protein micelles. Our results provide insight into the early steps of protein-mediated biomimetic nucleation of iron oxide and point to the importance of an extended protein surface during nanoparticle formation

Integrated Self-Assembly of the Mms6 Magnetosome Protein to Form an Iron-Responsive Structure

A common feature of biomineralization proteins is their self-assembly to produce a surface consistent in size with the inorganic crystals that they produce. Mms6, a small protein of 60 amino acids from Magnetospirillum magneticum strain AMB-1 that promotes the in vitro growth of superparamagnetic magnetite nanocrystals, assembles in aqueous solution to form spherical micelles that could be visualized by TEM and AFM. The results reported here are consistent with the view that the N and C-terminal domains interact with each other within one polypeptide chain and across protein units in the assembly. From studies to determine the amino acid residues important for self-assembly, we identified the unique GL repeat in the N-terminal domain with additional contributions from amino acids in other positions, throughout the molecule. Analysis by CD spectroscopy identified a structural change in the iron-binding C-terminal domain in the presence of Fe3+. A change in the intrinsic fluorescence of tryptophan in the N-terminal domain showed that this structural change is transmitted through the protein. Thus, self-assembly of Mms6 involves an interlaced structure of intra- and inter-molecular interactions that results in a coordinated structural change in the protein assembly with iron binding

Protein patterns template arrays of magnetic nanoparticles

Controlling the morphology of magnetic nanoparticles and their spatial arrangement is crucial for manipulating their functional properties. The commonly available inorganic processes for the synthesis of uniform magnetic nanoparticles typically require extreme reaction conditions such as high temperatures or harsh reagents, rendering them unsuitable for making functionalized magnetic nanoparticles with tunable properties controlled by biomolecules. Biomimetic procedures, inspired by the production of uniform magnetite and greigite crystals in magnetotactic bacteria, provide an alternative method, which can allow synthesis and spatial arrangement under ambient conditions. Mms6, an amphiphilic protein found in magnetosome membranes in Magnetospirillum magneticum strain AMB-1, can control the morphology of magnetite nanoparticles, both in vivo and in vitro. In this work, we have demonstrated the patterning of Mms6 and the formation of patterns of magnetic nanoparticles on selective regions of surfaces by directed self-assembly and control over surface chemistry, enabling facile spatial control in applications such as high density data storage and biosensors. Using microcontact printing we have obtained various patterns of 1-octadecane thiol (ODT) and protein resistant poly(ethylene glycol)methyl ether thiol (PEG) layers on gold surfaces. Atomic force microscopy (AFM) and fluorescence microscopy studies show the patterning of Mms6 on the ODT patterns and not on the PEG regions. Magnetic nanoparticles were grown on these surfaces by a co-precipitation method over immobilized protein. AFM and scanning electron microscopy (SEM) results show the localized growth of magnetic nanocrystals selectively on the Mms6 template, which in turn was determined by the ODT regions. Magnetic force measurements were conducted to assess the localization of magnetic nanoparticles on the pattern

Self-Assembly and Biphasic Iron-Binding Characteristics of Mms6, A Bacterial Protein That Promotes the Formation of Superparamagnetic Magnetite Nanoparticles of Uniform Size and Shape

Highly ordered mineralized structures created by living organisms are often hierarchical in structure with fundamental structural elements at nanometer scales. Proteins have been found responsible for forming many of these structures, but the mechanisms by which these biomineralization proteins function are generally poorly understood. To better understand its role in biomineralization, the magnetotactic bacterial protein, Mms6, which promotes the formation in vitro of superparamagnetic magnetite nanoparticles of uniform size and shape, was studied for its structure and function. Mms6 is shown to have two phases of iron binding: one high affinity and stoichiometric and the other low affinity, high capacity, and cooperative with respect to iron. The protein is amphipathic with a hydrophobic N-terminal domain and hydrophilic C-terminal domain. It self-assembles to form a micelle, with most particles consisting of 20–40 monomers, with the hydrophilic C-termini exposed on the outside. Studies of proteins with mutated C-terminal domains show that the C-terminal domain contributes to the stability of this multisubunit particle and binds iron by a mechanism that is sensitive to the arrangement of carboxyl/hydroxyl groups in this domain

Morphological Transformations in the Magnetite Biomineralizing Protein Mms6 in Iron Solutions: A Small-Angle X-ray Scattering Study

Magnetotactic bacteria that produce magnetic nanocrystals of uniform size and well-defined morphologies have inspired the use of biomineralization protein Mms6 to promote formation of uniform magnetic nanocrystals in vitro. Small angle X-ray scattering (SAXS) studies in physiological solutions reveal that Mms6 forms compact globular three-dimensional (3D) micelles (approximately 10 nm in diameter) that are, to a large extent, independent of concentration. In the presence of iron ions in the solutions, the general micellar morphology is preserved, however, with associations among micelles that are induced by iron ions. Compared with Mms6, the m2Mms6 mutant (with the sequence of hydroxyl/carboxyl containing residues in the C-terminal domain shuffled) exhibits subtle morphological changes in the presence of iron ions in solutions. The analysis of the SAXS data is consistent with a hierarchical core–corona micellar structure similar to that found in amphiphilic polymers. The addition of ferric and ferrous iron ions to the protein solution induces morphological changes in the micellar structure by transforming the 3D micelles into objects of reduced dimensionality of 2, with fractal-like characteristics (including Gaussian-chain-like) or, alternatively, platelet-like structures