This thesis describes a sequence of experiments which have been performed with the intention to produce synthetic nacre. Synthetic nacre is a biomimetic material based on nacre, a material which can be found in mollusc shells. Nacre is a nanocomposite which has improved mechanical properties compared to the principal component aragonite (95% wt%). The improved properties of nacre are derived from the polymeric components in the composite which allows from redistribution of forces under load. Carbonates sequester CO2 in the geological CO2-cycle. If precursor are sourced correctly, the CaCO3 in synthetic nacre can temporarily sequester CO2. Crystals with the intended pseudohexagonal morphology have been synthesised. However, subsequent quantitative analysis could not support these findings in a follow-up experiment. This discrepancy might have been caused by differences in the method. Moist nanopaper was found to be mineralisable while maintaining a layered structure.

Kieselalgen – auch Diatomeen genannt – verfügen über die einzigartige Fähigkeit, nanostrukturierte, hierarchisch aufgebaute Zellwände aus Siliziumdioxid – auch als Biosilica bekannt – mit beispielloser Genauigkeit und Reproduzierbarkeit zu bilden. Ein tieferes Verständnis für diesen Prozess, der als “Biomineralisation“ bekannt ist, ist nicht nur auf dem Gebiet der Grundlagenforschung zu Kieselalgen sehr bedeutsam, sondern auch für die Nutzung dieser Nanostrukturierung in den Materialwissenschaften oder der Nanobiotechnologie. Nach dem derzeitigem Stand der Wissenschaft wird diese Strukturierung durch die Selbstorganisation von Proteinmustern, an denen sich das Siliziumdioxid bildet, erreicht. Um die Funktion und das Zusammenspiel einzelner Proteine, die an diesem Biomineralisationsprozess beteiligt sind, entschlüsseln zu können, ist es essentiell ihre strukturelle Organisation aufzuklären und diese mit den morphologischen Zellwandmerkmalen zu korrelieren. Die Größenordnung dieser Merkmale ist im Bereich von Nanometern angesiedelt. Mit Hilfe der Elektronenmikroskopie können diese Biosilicastrukturen aufgelöst werden, jedoch ist keine proteinspezifische Information verfügbar. Ziel dieser Arbeit war es daher, eine Technik zu etablieren, die in der Lage ist, einzelne Biosilica-assozierte Proteine mit Nanometer-Präzision zu lokalisieren. Um dieses Ziel zu erreichen, wurde Einzelmoleküllokalisationsmikroskopie (single-molecule localization microscopy, kurz: SMLM) beispielhaft in der Kieselalge Thalassiosira pseudonana etabliert. Die Position verschiedener Biosilica-assoziierte Proteine innerhalb des Biosilicas und nach dessen chemischer Auflösung wurde mit einer hohen räumlichen Auflösung bestimmt. Um quantitative Ergebnisse zu erhalten, wurde ein Analyse-Workflow entwickelt, der grafische Benutzeroberflächen und Skripte für die Visualisierung, das Clustering und die Kolokalisation von SMLM Daten beinhaltet. Um optimale Markierungen für SMLM an Biosilica-eingebetteten Proteinen zu finden, wurde ein umfassendes Screening von photo-schaltbaren fluoreszierenden Proteinen durchgeführt. Diese wurden als Fusionsproteine mit Silaffin3, einem Protein, welches eng mit der Biosilica-Zellwand assoziiert ist, exprimiert. Es konnte gezeigt werden, dass nur drei von sechs Kandidaten funktional sind, wenn sie in Biosilica eingebettet sind. Silaffin3 konnte indirekt mittels SMLM mit einer Lokalisationsgenauigkeit von 25 nm detektiert werden. Dies erlaubte es, seine strukturelle Organisation aufzulösen und Silaffin3 als eine Hauptkomponente in der Basalkammer der Fultoportulae zu identifizieren. / Diatoms feature the unique ability to form nanopatterned hierarchical silica cell walls with unprecedented accuracy and reproducibility. Gathering a deeper understanding of this process that is known as “biomineralization” is vitally important not only in the field of diatom research. In fact, the nanopatterning can also be exploited in the fields of material sciences or nanobiotechnology. According to the current understanding, the self-assembly of protein patterns along which biosilica is formed is key to this nanopatterning. Thus, in order to unravel the function of individual proteins that are involved in this biomineralization process, their structural organization has to be deciphered and correlated to morphological cell wall features that are in the order of tens of nanometer. Electron microscopy is able to resolve these features but does not provide protein-specific information. Therefore, a technique has to be established that is able to localize individual biosilica-associated proteins with nanometer precision. To achieve this objective, single-molecule localization microscopy (SMLM) for the diatom Thalassiosira pseudonana has been pioneered and exploited to localize different biosilica associated proteins inside silica and after silica removal. To obtain quantitative data, an analysis workflow was developed including graphical user interfaces and scripts for SMLM visualization, clustering, and co-localization. In order to find optimal labels for SMLM to target biosilica-embedded proteins, a comprehensive screening of photo-controllable fluorescent proteins has been carried out. Only three of six candidates were functional when embedded inside biosilica and fused to Silaffin3 – a protein that is tightly associated with the biosilica cell wall. Silaffin3 could be localized using SMLM with a localization precision of 25 nm. This allowed to resolve its structural organization and therefore identified Silaffin3 as a major component in the basal chamber of the fultoportulae. Additionally, co-localization studies on cingulins – a protein family hypothesized to be involved in silica formation – have been performed to decipher their pattern-function relationship. Towards this end, novel imaging strategies, co-localization calculations and pattern quantifications have been established. With the help of these results, the spatial arrangement of cingulins W2 and Y2 could be compared with unprecedented resolution. In summary, this work has laid ground for quantitative SMLM studies of proteins in diatoms in general and contributed insights into the spatial organization of proteins involved in biomineralization in the diatom T. pseudonana.

Kieselalgen – auch Diatomeen genannt – verfügen über die einzigartige Fähigkeit, nanostrukturierte, hierarchisch aufgebaute Zellwände aus Siliziumdioxid – auch als Biosilica bekannt – mit beispielloser Genauigkeit und Reproduzierbarkeit zu bilden. Ein tieferes Verständnis für diesen Prozess, der als “Biomineralisation“ bekannt ist, ist nicht nur auf dem Gebiet der Grundlagenforschung zu Kieselalgen sehr bedeutsam, sondern auch für die Nutzung dieser Nanostrukturierung in den Materialwissenschaften oder der Nanobiotechnologie. Nach dem derzeitigem Stand der Wissenschaft wird diese Strukturierung durch die Selbstorganisation von Proteinmustern, an denen sich das Siliziumdioxid bildet, erreicht. Um die Funktion und das Zusammenspiel einzelner Proteine, die an diesem Biomineralisationsprozess beteiligt sind, entschlüsseln zu können, ist es essentiell ihre strukturelle Organisation aufzuklären und diese mit den morphologischen Zellwandmerkmalen zu korrelieren. Die Größenordnung dieser Merkmale ist im Bereich von Nanometern angesiedelt. Mit Hilfe der Elektronenmikroskopie können diese Biosilicastrukturen aufgelöst werden, jedoch ist keine proteinspezifische Information verfügbar. Ziel dieser Arbeit war es daher, eine Technik zu etablieren, die in der Lage ist, einzelne Biosilica-assozierte Proteine mit Nanometer-Präzision zu lokalisieren. Um dieses Ziel zu erreichen, wurde Einzelmoleküllokalisationsmikroskopie (single-molecule localization microscopy, kurz: SMLM) beispielhaft in der Kieselalge Thalassiosira pseudonana etabliert. Die Position verschiedener Biosilica-assoziierte Proteine innerhalb des Biosilicas und nach dessen chemischer Auflösung wurde mit einer hohen räumlichen Auflösung bestimmt. Um quantitative Ergebnisse zu erhalten, wurde ein Analyse-Workflow entwickelt, der grafische Benutzeroberflächen und Skripte für die Visualisierung, das Clustering und die Kolokalisation von SMLM Daten beinhaltet. Um optimale Markierungen für SMLM an Biosilica-eingebetteten Proteinen zu finden, wurde ein umfassendes Screening von photo-schaltbaren fluoreszierenden Proteinen durchgeführt. Diese wurden als Fusionsproteine mit Silaffin3, einem Protein, welches eng mit der Biosilica-Zellwand assoziiert ist, exprimiert. Es konnte gezeigt werden, dass nur drei von sechs Kandidaten funktional sind, wenn sie in Biosilica eingebettet sind. Silaffin3 konnte indirekt mittels SMLM mit einer Lokalisationsgenauigkeit von 25 nm detektiert werden. Dies erlaubte es, seine strukturelle Organisation aufzulösen und Silaffin3 als eine Hauptkomponente in der Basalkammer der Fultoportulae zu identifizieren.:1 INTRODUCTION 1 1.1 Diatoms – a model system for biomineralization 3 1.2 Imaging of biosilica and associated organic components 8 1.3 Single-molecule localization microscopy (SMLM) 10 2 METHODS & METHOD DEVELOPMENT FOR SMLM DATASETS 17 2.1 Super-resolution reconstruction 19 2.2 Tools for SMLM resolution estimates 21 2.3 Voronoi tessellation for noise-removal and cluster estimation 25 2.4 Tools for SMLM cluster analysis 27 2.5 Coordinate-based co-localization 32 2.6 PairRice – A novel algorithm to extract distances between cluster pairs 33 2.7 SiMoNa – A new GUI for exploring SMLM datasets 35 3 RESOLUTION OF THE SMLM SETUP TESTED WITH DNA ORIGAMI NANOSTRUCTURES 41 3.1 DNA origami as a length standard 42 3.2 Global resolution estimates 44 3.3 Local resolution estimates 47 3.4 Conclusion 53 4 EVALUATION OF PHOTO-CONTROLLABLE FLUORESCENT PROTEINS FOR PALM IN DIATOMS 55 4.1 Selecting PCFPs to minimize interference with the diatom autofluorescence 56 4.2 Screening results for cytosolic and biosilica-embedded PCFPs 58 4.3 The underlying conversion mechanism 61 4.4 Conclusion 63 5 IMAGING THE SIL3 MESHWORK 65 5.1 Analyzing protein layer thickness using tpSil3-Dendra2 65 5.2 Imaging the valve region using tpSil3 68 5.3 Resolution and localization parameters of tpSil3 70 5.4 Conclusion 72 6 DECIPHERING CINGULIN PATTERNS WITH CO LOCALIZATION STUDIES 73 6.1 A two-color cingulin construct for PALM-STORM 73 6.2 Steps towards PALM-STORM: screening, alignment, and imaging routine 76 6.3 Co-localization studies: quantification, clustering, and correlations 83 6.4 Conclusion 91 7 OUTLOOK 93 8 MATERIALS & METHODS 97 8.1 Microscope specifications 97 8.2 DNA origami annealing and AFM measurements 99 8.3 Diatom sample preparations 100 8.4 Fluorescence imaging conditions 102 8.5 Buffer systems 103 9 APPENDICES 105 9.1 Tables and Protocols 105 9.2 Satellite projects 112 9.2.1 Quantitative fluorescence intensity analysis of 3D time-lapse confocal microscopy data in diatoms 112 9.2.2 Applying neural networks to filter SMLM localizations 118 9.2.3 In vivo imaging at super-resolution conditions using SOFI 121 9.2.4 Quantifying chromatic aberrations in the microscope using fiducials 123 10 REFERENCES 127 / Diatoms feature the unique ability to form nanopatterned hierarchical silica cell walls with unprecedented accuracy and reproducibility. Gathering a deeper understanding of this process that is known as “biomineralization” is vitally important not only in the field of diatom research. In fact, the nanopatterning can also be exploited in the fields of material sciences or nanobiotechnology. According to the current understanding, the self-assembly of protein patterns along which biosilica is formed is key to this nanopatterning. Thus, in order to unravel the function of individual proteins that are involved in this biomineralization process, their structural organization has to be deciphered and correlated to morphological cell wall features that are in the order of tens of nanometer. Electron microscopy is able to resolve these features but does not provide protein-specific information. Therefore, a technique has to be established that is able to localize individual biosilica-associated proteins with nanometer precision. To achieve this objective, single-molecule localization microscopy (SMLM) for the diatom Thalassiosira pseudonana has been pioneered and exploited to localize different biosilica associated proteins inside silica and after silica removal. To obtain quantitative data, an analysis workflow was developed including graphical user interfaces and scripts for SMLM visualization, clustering, and co-localization. In order to find optimal labels for SMLM to target biosilica-embedded proteins, a comprehensive screening of photo-controllable fluorescent proteins has been carried out. Only three of six candidates were functional when embedded inside biosilica and fused to Silaffin3 – a protein that is tightly associated with the biosilica cell wall. Silaffin3 could be localized using SMLM with a localization precision of 25 nm. This allowed to resolve its structural organization and therefore identified Silaffin3 as a major component in the basal chamber of the fultoportulae. Additionally, co-localization studies on cingulins – a protein family hypothesized to be involved in silica formation – have been performed to decipher their pattern-function relationship. Towards this end, novel imaging strategies, co-localization calculations and pattern quantifications have been established. With the help of these results, the spatial arrangement of cingulins W2 and Y2 could be compared with unprecedented resolution. In summary, this work has laid ground for quantitative SMLM studies of proteins in diatoms in general and contributed insights into the spatial organization of proteins involved in biomineralization in the diatom T. pseudonana.:1 INTRODUCTION 1 1.1 Diatoms – a model system for biomineralization 3 1.2 Imaging of biosilica and associated organic components 8 1.3 Single-molecule localization microscopy (SMLM) 10 2 METHODS & METHOD DEVELOPMENT FOR SMLM DATASETS 17 2.1 Super-resolution reconstruction 19 2.2 Tools for SMLM resolution estimates 21 2.3 Voronoi tessellation for noise-removal and cluster estimation 25 2.4 Tools for SMLM cluster analysis 27 2.5 Coordinate-based co-localization 32 2.6 PairRice – A novel algorithm to extract distances between cluster pairs 33 2.7 SiMoNa – A new GUI for exploring SMLM datasets 35 3 RESOLUTION OF THE SMLM SETUP TESTED WITH DNA ORIGAMI NANOSTRUCTURES 41 3.1 DNA origami as a length standard 42 3.2 Global resolution estimates 44 3.3 Local resolution estimates 47 3.4 Conclusion 53 4 EVALUATION OF PHOTO-CONTROLLABLE FLUORESCENT PROTEINS FOR PALM IN DIATOMS 55 4.1 Selecting PCFPs to minimize interference with the diatom autofluorescence 56 4.2 Screening results for cytosolic and biosilica-embedded PCFPs 58 4.3 The underlying conversion mechanism 61 4.4 Conclusion 63 5 IMAGING THE SIL3 MESHWORK 65 5.1 Analyzing protein layer thickness using tpSil3-Dendra2 65 5.2 Imaging the valve region using tpSil3 68 5.3 Resolution and localization parameters of tpSil3 70 5.4 Conclusion 72 6 DECIPHERING CINGULIN PATTERNS WITH CO LOCALIZATION STUDIES 73 6.1 A two-color cingulin construct for PALM-STORM 73 6.2 Steps towards PALM-STORM: screening, alignment, and imaging routine 76 6.3 Co-localization studies: quantification, clustering, and correlations 83 6.4 Conclusion 91 7 OUTLOOK 93 8 MATERIALS & METHODS 97 8.1 Microscope specifications 97 8.2 DNA origami annealing and AFM measurements 99 8.3 Diatom sample preparations 100 8.4 Fluorescence imaging conditions 102 8.5 Buffer systems 103 9 APPENDICES 105 9.1 Tables and Protocols 105 9.2 Satellite projects 112 9.2.1 Quantitative fluorescence intensity analysis of 3D time-lapse confocal microscopy data in diatoms 112 9.2.2 Applying neural networks to filter SMLM localizations 118 9.2.3 In vivo imaging at super-resolution conditions using SOFI 121 9.2.4 Quantifying chromatic aberrations in the microscope using fiducials 123 10 REFERENCES 127

With respect to the principles of biomineralization, it is of interest to study the crystallization of calcium oxalates under various experimental conditions. Calcium oxalates play decisive roles as biominerals in plants and as pathological “urinary/kidney stones” in vertebrates. Calcium oxalate exists in three different hydration states; calcium oxalate monohydrate (COM, monoclinic, a = 6.290(1)Å, b = 14.583(1)Å, c = 10.116(1)Å, β = 109.46°, P21/c), calcium oxalate dihydrate (COD, tetragonal, a = b = 12.371(3)Å, c = 7.357(2)Å, α = β = γ = 90°, I4/m) and calcium oxalate trihydrate (COT, triclinic, a = 6.11(1)Å, b = 7.167(2)Å, c = 8.457(2)Å, α = 76.5(2)°, β = 70.35(2)°, γ = 70.62(2)°, P ). Monoclinic COM and tetragonal COD are the most common phyto-crystals and the main constituents of kidney and urinary stones. The occurrence of calcium oxalates in plants represents a useful biogenesis (protection against herbivores) unlike the devastating occurrence in renal tubules. Therefore, biomineralization can be physiological or pathological. A systematic investigation of the morphological evolution of calcium oxalates in the presence of organic components is essential for understanding the mechanism of “pathological biomineralization”. In order to understand the pathological biomineralization of uroliths, it is necessary grow calcium oxalates comparable in morphology under similar growth conditions. The formation of calcium oxalate stones within a gelatinous state of proteins, polysaccharides, lipids and other biomacromolecules under a flow of supersaturated urine supports the fact that an “organic” gel model can simulate the process of urinary stone formation under in vitro conditions. Furthermore, synthetic polymers with precisely known functions and solution behaviours are better choices to understand the interaction of acidic proteins with calcium oxalates. Therefore, as a first step to unravel the complex pathology of uro/nephro lithiasis, we started to examine the structure and morphology of calcium oxalates crystallized in the presence of organic additives such as the sodium salt of polyacrylic acid (PAA) as well as agar gel. The influence of initial calcium oxalate concentration, pH and concentration of the additives on the formation of hydration states of calcium oxalates have been investigated along with the stated general methods. Apart from the three hydrated forms, calcium oxalate exists also in the anhydrous form (COA). Although three modifications of COA (α, β and γ) are reported in the literatures, the crystal structures and phase transformations were controversially discussed. We have been able to reveal the crystal structure of the β-modification of the anhydrous calcium oxalate by a combination of atomistic simulations and Rietveld refinements on the basis of powder X-ray diffraction pattern. β-COA belongs to the monoclinic system with unit cell parameters, a = 6.1644(3)Å, b = 7.3623(2)Å, c = 9.5371(5)Å, β = 90.24(2)°, P2/m (No. 10). The dehydration of COM was mimicked in silico to receive an initial model of the crystal structure of anhydrous calcium oxalate. This general approach may also be accessible for other decomposition processes ending up with crystalline powders of unknown crystal structure. No evidence for transformations from or to the α- or γ- modifications was found during our investigations. The growth pattern of COD crystals precipitated from aqueous solutions in the presence of PAA is clearly dependent on the concentration of PAA. By increasing the concentration of PAA, the shape of COD has been found to change from tetragonal bi-pyramids with dominant (101) pyramidal faces to tetragonal prisms with dominant (100) prism faces and finally to dumbbells. At still higher PAA concentrations, the morphology is reverted back to rod-like tetragonal prisms. Apart from these experiments, the interaction of PAA with (100) and (101) crystal faces of COD was explored with the aid of atomistic simulations. The simulation confirmed that during the development of the aggregates, strong interactions of PAA with the (100) faces take over control of morphologies. Our investigations show that the inner architecture of all the morphological varieties of COD was found to be dominated by an inner “core” consisting of thin elongated crystallites together with incorporated PAA and an outer “shell” formed as a consequence of secondary nucleation processes. We propose that for all types of COD aggregates, relative proportion of calcium oxalate and PAA dictates the shape and formation of nanometer sized crystallites which then aggregate and align to form the core. Such cores enriched with PAA may act as the sites for secondary nucleation events of calcium oxalate crystallites which then cover the core like a shell. In vitro experimental models for the growth of calcium oxalates can give valuable information on the growth and aggregation of urinary stones. Therefore, the “double diffusion technique” in agar gel matrix has been used for the biomimetic growth of calcium oxalate (COM) stones. A great variety of morphological forms of COM are produced in agar gel matrices (2 wt.-% agar gel of pH 8.5) ranging from platy crystallites to dumbbells and spherulites. The COM dumbbells and spherulites are assumed to be formed by the aggregation of smaller crystallites as a consequence of increased supersaturation inside the gel. Moreover, an increase of the pH value of the agar gel has been found to suppress the growth of COM and favours the growth of COD. The morphology of COD crystals grown in 2 wt.-% agar gel of pH 11.5 includes tetragonal prisms and dumbbells. The system calcium oxalate/ PAA/ H2O is a suitable model system for the investigation of principles of biomineral growth (shape development) in general. Our results demonstrate that the double diffusion technique in agar gel is a convenient route to grow calcium oxalate aggregates showing close resemblance to biogenic calculi and to study their ontogeny.

No description available.

Biological materials have ever been used by humans because of their remarkable properties. This is surprising since the materials are formed under physiological conditions and with commonplace constituents. Nature thus not only provides us with inspiration for designing new materials but also teaches us how to use soft molecules to tune interparticle and external forces to structure and assemble simple building blocks into functional entities. Magnetotactic bacteria and their chain of magnetosomes represent a striking example of such an accomplishment where a very simple living organism controls the properties of inorganics via organics at the nanometer-scale to form a single magnetic dipole that orients the cell in the Earth magnetic field lines. My group has developed a biological and a bio-inspired research based on these bacteria. My research, at the interface between chemistry, materials science, physics, and biology focuses on how biological systems synthesize, organize and use minerals. We apply the design principles to sustainably form hierarchical materials with controlled properties that can be used e.g. as magnetically directed nanodevices towards applications in sensing, actuating, and transport. In this thesis, I thus first present how magnetotactic bacteria intracellularly form magnetosomes and assemble them in chains. I developed an assay, where cells can be switched from magnetic to non-magnetic states. This enabled to study the dynamics of magnetosome and magnetosome chain formation. We found that the magnetosomes nucleate within minutes whereas chains assembles within hours. Magnetosome formation necessitates iron uptake as ferrous or ferric ions. The transport of the ions within the cell leads to the formation of a ferritin-like intermediate, which subsequently is transported and transformed within the magnetosome organelle in a ferrihydrite-like precursor. Finally, magnetite crystals nucleate and grow toward their mature dimension. In addition, I show that the magnetosome assembly displays hierarchically ordered nano- and microstructures over several levels, enabling the coordinated alignment and motility of entire populations of cells. The magnetosomes are indeed composed of structurally pure magnetite. The organelles are partly composed of proteins, which role is crucial for the properties of the magnetosomes. As an example, we showed how the protein MmsF is involved in the control of magnetosome size and morphology. We have further shown by 2D X-ray diffraction that the magnetosome particles are aligned along the same direction in the magnetosome chain. We then show how magnetic properties of the nascent magnetosome influence the alignment of the particles, and how the proteins MamJ and MamK coordinate this assembly. We propose a theoretical approach, which suggests that biological forces are more important than physical ones for the chain formation. All these studies thus show how magnetosome formation and organization are under strict biological control, which is associated with unprecedented material properties. Finally, we show that the magnetosome chain enables the cells to find their preferred oxygen conditions if the magnetic field is present. The synthetic part of this work shows how the understanding of the design principles of magnetosome formation enabled me to perform biomimetic synthesis of magnetite particles within the highly desired size range of 25 to 100 nm. Nucleation and growth of such particles are based on aggregation of iron colloids termed primary particles as imaged by cryo-high resolution TEM. I show how additives influence magnetite formation and properties. In particular, MamP, a so-called magnetochrome proteins involved in the magnetosome formation in vivo, enables the in vitro formation of magnetite nanoparticles exclusively from ferrous iron by controlling the redox state of the process. Negatively charged additives, such as MamJ, retard magnetite nucleation in vitro, probably by interacting with the iron ions. Other additives such as e.g. polyarginine can be used to control the colloidal stability of stable-single domain sized nanoparticles. Finally, I show how we can “glue” magnetic nanoparticles to form propellers that can be actuated and swim with the help of external magnetic fields. We propose a simple theory to explain the observed movement. We can use the theoretical framework to design experimental conditions to sort out the propellers depending on their size and effectively confirm this prediction experimentally. Thereby, we could image propellers with size down to 290 nm in their longer dimension, much smaller than what perform so far. / Biologische Materialien wie Knochen, Muscheln und Holz wurden von den Menschen seit den ältesten Zeiten verwendet. Diese biologisch gebildeten Materialien haben bemerkenswerte Eigenschaften. Dies ist besonders überraschend, da sie unter physiologischen Bedingungen und mit alltäglichen Bestandteilen gebildet sind. Die Natur liefert uns also nicht nur mit Inspiration für die Entwicklung neuer Materialien, sondern lehrt uns auch, wie biologische Additiven benutzen werden können, um einfache synthetische Bausteine in funktionale Einheiten zu strukturieren. Magnetotaktischen Bakterien und ihre Kette von Magnetosomen sind ein Beispiel, wo einfache Lebewesen die Eigenschaften von anorganischen Materialien steuern, um sich entlang den magnetischen Feldlinien der Erde zu orientieren. Die von den Bakterien gebildeten Magnetosomen sind von besonderem Interesse, da mit magnetischen Eisenoxid-Nanopartikeln in den letzten zehn Jahren einer Vielzahl von Bio-und nanotechnologischen Anwendungen entwickelt worden sind. In dieser Arbeit stelle ich eine biologische und eine bio-inspirierte Forschung auf der Grundlage der magnetotaktischen Bakterien vor. Diese Forschung verbindet die neuesten Entwicklungen von Nanotechnik in der chemischen Wissenschaft, die neuesten Fortschritte der Molekularbiologie zusammen mit modernen Messverfahren. Mein Forschungsschwerpunkt liegt somit an der Schnittstelle zwischen Chemie, Materialwissenschaften, Physik und Biologie. Ich will verstehen, wie biologische Systeme Materialien synthetisieren und organisieren, um Design-Prinzipien zu extrahieren, damit hierarchischen Materialien mit kontrollierten Eigenschaften nachhaltig gebildet werden.

L’étude présentée dans ce manuscrit porte sur la réplication de structures naturelles multi-échelles et multifonctionnelles, que sont les ailes des papillons Morpho rhetenor, Morpho menelaus et Papilio ulysse, ou celles de la cigale Cicada orni. De telles structures sont en effet constituées de sous-structures à différentes échelles, du centimètre au nanomètre, et à chacune de ces échelles est associée une propriété ou fonction. On parle alors de multifonctionnalité. Cet atout, très recherché actuellement pour nos futurs objets et matériaux, est accessible par deux voies de complexification : celle de la composition chimique du ou des matériaux constituant l’objet (matériaux composites, hybrides organique-inorganique) et/ou celle de leur géométrie (structuration). Or, si nos connaissances en chimie nous permettent de mettre en œuvre la première voie, l’élaboration de structures multi-échelles est encore difficile par nos techniques actuelles de structuration (lithographie par exemple). Ainsi, pour augmenter les propriétés d’un système possédant une géométrie multi-échelles existante dans la nature, nous avons réalisé des répliques des structures naturelles précédemment évoquées dans des matériaux inorganiques (TiO2 et SiO2), soit des matériaux très différents du complexe chitino-protéique qui constituent les ailes organiques. A cette fin, trois méthodes ont été utilisées : un dépôt de matière dans les structures naturelles par voie sol-gel, un dépôt par pulvérisation cathodique et une minéralisation directe de la structure des ailes, s’inspirant de processus de biominéralisation. / The present study deals with the replication of multiscale and multifunctional natural structures. These natural structures are wings of Morpho rhetenor, Morpho menelaus and Papilio ulysse butterflies, and those of Cicada orni cicada. Such structures are composed of smaller structures at different scales, from centimetre to nanometre, and to each of these scales is associated a property or a function. This we call multifunctionality. This multifunctionality is expected to become a property of our future objects or materials, and can be achieved by two different ways: to make the material(s) chemical composition of the object more complex (composite, hybrid organic-inorganic materials) and/or to make its architecture more complex (structuration). Although it is possible to achieve the first (chemical composition), we have so far been unable to successfully make multiscale structures with our current structuration techniques (lithography for example). Therefore, to increase the properties of a system characterised by a multiscale structure seen in nature, we have made replicas of the natural structures previously presented in inorganic materials (TiO2 and SiO2). That is to say, very different materials in comparison with the natural chitin-protein complex. To do this, three methods were used: a sol-gel solution deposition in the natural structures, a physical vapor deposition and a direct mineralization of the wings structure, which is inspired by natural biomineralization processes.

In dieser Arbeit ist es gelungen, die Bedeutung physikalisch-chemischer Mechanismen in der Biomineralisation gegenüber der bisher angenommenen Dominanz spezifischer biomolekularer Erkennungsmechanismen aufzuzeigen. Dazu wurden drei Ansätze verfolgt: Zum einen wurden Studien zur Calciumcarbonatkristallisation durchgeführt. Zum anderen wurde das Biomineral Perlmutt intensiv untersucht. Als drittes wurde ein Modellsystem entwickelt, mit dem künstliches Perlmutt synthetisiert und ein Mechanismus für die Perlmuttmineralisation vorgeschlagen werden konnte. <br><br> Im ersten Schritt wurden in einem simplen Kristallisationsansatz komplexe Calciumcarbonatüberstrukturen ohne die Verwendung von Additiven synthetisiert. Es wurde gezeigt, daß diese durch orientierte Anlagerung von Nanopartikeln gebildet werden, bei der dipolare Felder eine wichtige Rolle zu spielen scheinen. Dieser Mechansimus war bislang für Calciumcarbonat unbekannt und ermöglicht die Synthese komplexer Kristallmorphologien, wodurch die Frage aufgeworfen wird, ob er bei der Biomineralbildung von Bedeutung sein kann. Durch Einsatz minimaler Mengen eines einfachen, synthetischen Additivs bei der Kristallisation wurden zu Überstrukturen angeordnete Aragonitplättchen synthetisiert, die von einer wenige nm dicken Schicht aus amorphen Calciumcarbonat umgeben sind. Eine solche Schicht wurde auch bei den Aragonitplättchen Perlmutts entdeckt (s.u.) und bietet möglicherweise in verschiedenen Systemen eine Erklärung für die Stabilisierung der sonst metastabilen Aragonitphase. <br><br> Im zweiten Schritt wurden bei der Untersuchung von natürlichem Perlmutt zwei bislang unbekannte Strukturmerkmale entdeckt: Es gibt Bereiche, die nicht aus den charakteristischen Plättchen bestehen, sondern wesentlich weniger stark mineralisert sind. Die Mineralphase besteht in diesen Bereichen aus Nanopartikeln. Es wurde weiterhin gezeigt, daß die Aragonitplättchen von einer wenige nm dicken Schicht aus amorphem Calciumcarbonat umgeben ist. Die gängigen Modelle der Perlmuttbildung sind mit diesen Beobachtungen nicht zu vereinbaren und somit zu hinterfragen. Dagegen deuten diese Ergebnisse ein Wachstum von Perlmutt über ACC-Nanopartikel an. <br><br> Unter der Annahme der Bedeutung physikalisch-chemischer Mechanismen in der Biomineralisation wurde schließlich als dritter Schritt ein Ansatz zur in vitro-Retrosynthese von Biomineralien ausgehend von ihrer unlöslichen Matrix entwickelt. <br><br> Mit diesem Ansatz ist es erstmals gelungen, künstliches Perlmutt auf synthetischem Wege herzustellen, das morphologisch nicht vom Original zu unterscheiden ist. Die existierenden Unterschiede konnten zeigen, daß der Mineralisationsprozeß nicht auf ein spezifisches Mikroumgebungssystem beschränkt, sondern "allgemeiner gültig"' sein muß. <br><br> Bei der Retrosynthese gibt es zwei Schlüsselfaktoren: Zum einen die demineralisierte unlösliche Perlmuttmatrix als dreidimensionales Gerüst für das künstliche Perlmutt, zum anderen amorphe Precursorpartikel, die die Mineralphase bilden. Es werden keinerlei Proteine oder andere Biomoleküle verwendet. Dieser Ansatz bietet die Möglichkeit, den Mineralisationsprozeß an einem in vitro-Modellsystem zu verfolgen, was für das in vivo-System, wenn überhaupt, nur unter starken Einschränkungen möglich ist.<br><br> Es wurde gezeigt, daß das künstliche Perlmutt über die Mesoskalentransformation von ACC-Precursorn innerhalb der Matrix gebildet wird und als möglicher Mechanismus bei der Biomineralisation von natürlichem Perlmutt diskutiert. Es konnte in der vorliegenden Arbeit konsequent gezeigt werden, daß die Imitation von Biomineralisationsprozessen in in vitro-Ansätzen möglich ist, wobei chemisch-physikalische Parameter dominieren. <br><br> In zukünftigen Studien sollten einerseits die mechanischen Eigenschaften des künstlichen Perlmutts untersucht werden, wofür sich in Vorversuchen im Rahmen dieser Arbeit die Nanoindentierung als geeignet herausgestellt hat. Es sollte geprüft werden, ob das hier ermittelte Prinzip zur Mineralisierung in der Materialentwicklung angewendet werden kann. Andererseits sollte die Retrosynthese auf andere Systeme ausgeweitet und in vivo-Studien durchgeführt werden, um die Gültigkeit der vorgeschlagenen Mechanismen zu überprüfen. / This thesis highlights the importance of physical-chemical mechanisms in biomineralisation and, thus, challenges the widely accepted dominance of specific biomolecular recognition mechanisms. <br><br> The work is divided into three parts: the first part addresses the crystallisation of calcium carbonate; the second part focuses on an intensive study of the biomineral, nacre, and, lastly, a retrosynthesis model system is designed and applied to synthesize artificial nacre. A mechanism for nacre mineralisation in nature is proposed. <br><br> Initially, complex calcium carbonate superstructures were synthesized in the absence of any additive. These were shown to grow by an oriented attachment mechanism of nanoparticles, presumably under the influence of dipolar fields. This growth mechanism has, to date, not been described for calcium carbonate. This mechanism opens the possibility to synthesize complex crystal morphologies of calcium carbonate and arises the question as to whether it plays a role in the growth of biominerals. <br><br> With the presence of small amounts of additives in calcium carbonate crystallisation it was possible to synthesize superstructures of aragonite platelets, each of which surrounded by a layer of amorphous calcium carbonate (ACC). Such ACC layers were also found in natural nacre (see below) and may explain the stabilisation of the metastable calcium carbonate polymorph aragonite. <br><br> In the second part of this thesis two unknown features of nacre structures were distinguished: Some areas within the nacre do not consist of the characteristic aragonite platelets but are mineralized only to a low degree. In these areas the mineral phase is clearly composed of nanoparticles. Furthermore, the aragonite platelets of nacre are shown to be surrounded by an ACC layer. Both observations contradict the classical models of nacre growth mechanisms but hint towards a growth via ACC nanoparticles. <br><br> Assuming the importance of physical-chemical mechanisms in biomineralisation, an approach for the in vitro retrosynthesis of biominerals was designed. Through this, it was possible, for the first time, to synthesize artificial nacre, which was indistinguishable in morphology from the original. The non-morphological differences between original and synthesized nacre showed that the biological process of mineralization is not limited to one specific microenvironment, but must be more general. <br><br> Two key factors are of importance for the retrosynthesis approach: 1) The demineralised nacre matrix, which forms a scaffold for the artificial mineral phase and; 2) amorphous nanoparticles as precursors, which transform into the mineral phase. No proteins or other biomolecules were utilized. In this way, the biomineralisation process could be followed in an in vitro model, a process, which is hardly possible in such detail under in vivo conditions. This work proves that the artificial nacre grows by a mesoscale transformation of ACC nanoparticles, and discusses this mechanism as a possible growth mechanism of natural nacre. This work consequently shows that it is possible to imitate biomineralisation processes in vitro and that, in–vitro, these processes are driven by physico-chemical parameters. <br><br> Future studies will involve investigation of the mechanical properties of the artificial nacre. First experiments indicate, that nanoindentation is hereby suitable. The potential application of the in vitro mineralization mechanism for new material development will be investigated. Furthermore, the retrosynthesis will be applied to other biomineral systems and, subsequently, in vivo studies will be performed so as to investigate the role of the proposed mechanisms for the natural biomineralisation process.

Die Schwingungsspektroskopie, besonders die Raman-Spektroskopie, stellt ein wichtiges Werkzeug für Untersuchungen von Biomineralien dar. Raman-Spektroskopie wurde zur Untersuchung der organischen und anorganischen Bestandteile von Schwammskeletten eingesetzt. Die Raman-Spektroskopie trug auch zur Charakterisierung von biomimetischen Silikat-Präzipitaten bei. Durch ortsaufgelöste Raman-Spektroskopie konnte erstmalig die Verteilung von organischem Material in den extrahierten Silikatzellwänden von Kieselalgen nachgewiesen werden. Die ortsaufgelöste Raman-Spektroskopie wurde ebenfalls zur Untersuchung des SERS-Effekts an Zellwänden von Kieselalgen an die Silber-Nanopartikel gekoppelt waren eingesetzt.

[Truncated abstract] Clinical magnetic resonance imaging (MRI) scanners were used to investigate the measurement and imaging of ferrimagnetic particle concentrations in biological tissues in vivo. The presence of ferrimagnetic particles tends to increase the proton transverse relaxation rate (R2) of water protons in tissue. A quantitative image of R2 can be generated using a series of single spin echo magnetic resonance images acquired using clinical MRI scanners and analysing the images using techniques based on that reported by Clark and St. Pierre (2000). If ferrimagnetic particles have a high enough concentration, there is a monotonic relationship between particle concentration and R2; therefore an image of R2 gives a map of the ferrimagnetic particle concentration in the tissue. These techniques were used to investigate the feasibility of in vivo measurement of the concentration and distribution of both synthetic and biogenic ferrimagnetic particles in tissue. Rabbit liver was loaded with ferrimagnetic particles of ?-Fe2O3 (designed for magnetic hyperthermia treatment of liver tumours) by injecting various doses of a suspension of the particles into the hepatic artery in vivo. R2 images of the livers in vivo, excised, and dissected were generated from a series of single spin-echo images. Mean R2 values for samples of ferrimagnetic-particle-loaded liver dissected into approximate 1 cm cubes were found to linearly correlate with tissue iron concentration over the range from approximately 0.1 to at least 2.7 mg Fe/g dry tissue when measured at room temperature. Changing the temperature of ferrimagnetic-particle-loaded samples of liver from 1?C to 37?C had no observable effect on tissue R2 values. However, a small but significant decrease in R2 was found for control samples containing no ferrimagnetic material on raising the temperature from 1?C to 37?C. Both chemically measured iron ii concentrations and mean R2 values for rabbit livers with implanted tumours tended to be higher than those measured for tumour-free liver. This study indicates that tissue R2 measurement and imaging by nuclear magnetic resonance may have a useful role in magnetic hyperthermia therapy protocols for the treatment of liver cancer. In order to investigate the use of clinical MRI scanners to measure biogenic ferrimagnetic particle concentrations in human brain tissue, agar gel based phantoms containing ferrimagnetic particles were made in order to determine the lower concentration detection limit for such particles in a homogenous medium. Magnetite/maghemite nanoparticles were synthesized in the presence of either dextran or polyvinyl alcohol, yielding cluster- and necklace-like aggregates, respectively. Magnetization, Mossbauer spectroscopy, and microscopy measurements indicated that the arrangement of the particles within the aggregates affects the magnetic properties of the particles resulting in smaller particles in the clusters having higher superparamagnetic blocking temperatures than larger particles in the necklaces.