Sensing Layer for Ni Detection in Water Created by Immobilization of Bioengineered Flagellar Nanotubes on Gold Surfaces.

The environmental monitoring of Ni is targeted at a threshold limit value of 0.34 µM, as set by the World Health Organization. This sensitivity target can usually only be met by time-consuming and expensive laboratory measurements. There is a need for inexpensive, field-applicable methods, even if they are only used for signaling the necessity of a more accurate laboratory investigation. In this work, bioengineered, protein-based sensing layers were developed for Ni detection in water. Two bacterial Ni-binding flagellin variants were fabricated using genetic engineering, and their applicability as Ni-sensitive biochip coatings was tested. Nanotubes of mutant flagellins were built by in vitro polymerization. A large surface density of the nanotubes on the sensor surface was achieved by covalent immobilization chemistry based on a dithiobis(succimidyl propionate) cross-linking method. The formation and density of the sensing layer was monitored and verified by spectroscopic ellipsometry and atomic force microscopy. Cyclic voltammetry (CV) measurements revealed a Ni sensitivity below 1 µM. It was also shown that, even after two months of storage, the used sensors can be regenerated and reused by rinsing in a 10 mM solution of ethylenediaminetetraacetic acid at room temperature.

Biotemplated synthesis of magnetic filaments.

With the aim of creating one-dimensional magnetic nanostructures, we genetically engineered flagellar filaments produced by Salmonella bacteria to display iron- or magnetite-binding sites, and used the mutant filaments as templates for both nucleation and attachment of the magnetic iron oxide magnetite. Although nucleation from solution and attachment of nanoparticles to a pre-existing surface are two different processes, non-classical crystal nucleation pathways have been increasingly recognized in biological systems, and in many cases nucleation and particle attachment cannot be clearly distinguished. In this study we tested the magnetite-nucleating ability of four types of mutant flagella previously shown to be efficient binders of magnetite nanoparticles, and we used two other mutant flagella that were engineered to periodically display known iron-binding oligopeptides on their surfaces. All mutant filaments were demonstrated to be efficient as templates for the synthesis of one-dimensional magnetic nanostructures under ambient conditions. Both approaches resulted in similar final products, with randomly oriented magnetite nanoparticles partially covering the filamentous biological templates. In an external magnetic field, the viscosity of a suspension of the produced magnetic filaments showed a twofold increase relative to the control sample. The results of magnetic susceptibility measurements were also consistent with the magnetic nanoparticles occurring in linear structures. Our study demonstrates that biological templating can be used to produce one-dimensional magnetic nanostructures under benign conditions, and that modified flagellar filaments can be used for creating model systems in which crystal nucleation from solution can be experimentally studied.

Single crystalline superstructured stable single domain magnetite nanoparticles.

Magnetite nanoparticles exhibit magnetic properties that are size and organization dependent and, for applications that rely on their magnetic state, they usually have to be monodisperse. Forming such particles, however, has remained a challenge. Here, we synthesize 40 nm particles of magnetite in the presence of polyarginine and show that they are composed of 10 nm building blocks, yet diffract like single crystals. We use both bulk magnetic measurements and magnetic induction maps recorded from individual particles using off-axis electron holography to show that each 40 nm particle typically contains a single magnetic domain. The magnetic state is therefore determined primarily by the size of the superstructure and not by the sizes of the constituent sub-units. Our results fundamentally demonstrate the structure - property relationship in a magnetic mesoparticle.

Magnetite-Binding Flagellar Filaments Displaying the MamI Loop Motif.

This work aimed at developing a novel method for fabricating 1 D magnetite nanostructures with the help of mutated flagellar filaments. We constructed four different flagellin mutants displaying magnetite-binding motifs: two contained fragments of magnetosome-associated proteins from magnetotactic bacteria (MamI and Mms6), and synthetic sequences were used for the other two. A magnetic selection method identified the MamI mutant as having the highest binding affinity to magnetite. Filaments built from MamI loop-containing flagellin subunits were used as templates to form chains of magnetite nanoparticles along the filament by capturing them from suspension. Our study represents a proof-of-concept that flagellar filaments can be engineered to facilitate formation of 1 D magnetite nanostructures under ambient conditions. In addition, it proves the interaction between MamI and magnetite, with implications for the role of this protein in magnetotactic bacteria.

Genetic dissection of the mamAB and mms6 operons reveals a gene set essential for magnetosome biogenesis in Magnetospirillum gryphiswaldense.

Biosynthesis of bacterial magnetosomes, which are intracellular membrane-enclosed, nanosized magnetic crystals, is controlled by a set of >30 specific genes. In Magnetospirillum gryphiswaldense, these are clustered mostly within a large conserved genomic magnetosome island (MAI) comprising the mms6, mamGFDC, mamAB, and mamXY operons. Here, we demonstrate that the five previously uncharacterized genes of the mms6 operon have crucial functions in the regulation of magnetosome biomineralization that partially overlap MamF and other proteins encoded by the adjacent mamGFDC operon. While all other deletions resulted in size reduction, elimination of either mms36 or mms48 caused the synthesis of magnetite crystals larger than those in the wild type (WT). Whereas the mms6 operon encodes accessory factors for crystal maturation, the large mamAB operon contains several essential and nonessential genes involved in various other steps of magnetosome biosynthesis, as shown by single deletions of all mamAB genes. While single deletions of mamL, -P, -Q, -R, -B, -S, -T, and -U showed phenotypes similar to those of their orthologs in a previous study in the related M. magneticum, we found mamI and mamN to be not required for at least rudimentary iron biomineralization in M. gryphiswaldense. Thus, only mamE, -L, -M, -O, -Q, and -B were essential for formation of magnetite, whereas a mamI mutant still biomineralized tiny particles which, however, consisted of the nonmagnetic iron oxide hematite, as shown by high-resolution transmission electron microscopy (HRTEM) and the X-ray absorption near-edge structure (XANES). Based on this and previous studies, we propose an extended model for magnetosome biosynthesis in M. gryphiswaldense.

Biosynthesis of magnetic nanostructures in a foreign organism by transfer of bacterial magnetosome gene clusters.

The synthetic production of monodisperse single magnetic domain nanoparticles at ambient temperature is challenging. In nature, magnetosomes--membrane-bound magnetic nanocrystals with unprecedented magnetic properties--can be biomineralized by magnetotactic bacteria. However, these microbes are difficult to handle. Expression of the underlying biosynthetic pathway from these fastidious microorganisms within other organisms could therefore greatly expand their nanotechnological and biomedical applications. So far, this has been hindered by the structural and genetic complexity of the magnetosome organelle and insufficient knowledge of the biosynthetic functions involved. Here, we show that the ability to biomineralize highly ordered magnetic nanostructures can be transferred to a foreign recipient. Expression of a minimal set of genes from the magnetotactic bacterium Magnetospirillum gryphiswaldense resulted in magnetosome biosynthesis within the photosynthetic model organism Rhodospirillum rubrum. Our findings will enable the sustainable production of tailored magnetic nanostructures in biotechnologically relevant hosts and represent a step towards the endogenous magnetization of various organisms by synthetic biology.

Keeping Nanoparticles Fully Functional: Long-Term Storage and Alteration of Magnetite.

Magnetite is an iron oxide found in rocks. Its magnetic properties are used for paleoclimatic reconstructions. It can also be synthesized in the laboratory to exploit its magnetic properties for bio- and nanotechnological applications. However, although the magnetic properties depend on particle size in a well-understood manner, they also depend on the structure of the oxide, because magnetite oxidizes to maghemite under environmental conditions. The dynamics of this process have not been well described. Here, a study of the alteration of magnetite particles of different sizes as a function of their storage conditions is presented. Smaller nanoparticles are shown to oxidize more rapidly than larger ones, and that the lower the storage temperature, the lower the measured oxidation. In addition, the magnetic properties of the altered particles are not decreased dramatically, thus suggesting that this alteration will not impact the use of such nanoparticles as medical carriers.