High Aspect Ratio-Nanostructured Surfaces as Biological Metamaterials

Materials patterned with high-aspect-ratio nanostructures have features on similar lengthscales to cellular components. These surfaces are an extreme topography on the cellular level and have become useful tools for perturbing and sensing the cellular environment. Motivation comes from the ability of high-aspect-ratio nanostructures to deliver cargoes into cells and tissues, access the intracellular environment, and control cell behavior. These structures directly perturb cells’ ability to sense and respond to external forces, influencing cell fate and enabling new mechanistic studies. Through careful design of their nanoscale structure, these systems act as biological metamaterials, eliciting unusual biological responses. While predominantly used to interface eukaryotic cells, there is growing interest in non-animal and prokaryotic cell interfacing. Both experimental and theoretical studies have attempted to develop a mechanistic understanding for the observed behaviors, predominantly focusing on the cell – nanostructure interface. Here, we consider how high-aspect-ratio nanostructured surfaces are used to both stimulate and sense biological systems and discuss remaining research questions

Fabrication and development of high-aspect-ratio nanostructures for biointerfacing

This thesis describes the optimisation of the fabrication protocols for vertically-aligned nanostructures to be interfaced with living cells. The dissertation is organised around four aims. The first aim describes the optimisation of a nanoneedle fabrication process. Nanoneedles are regular arrays of conical mesoporous-silicon nanostructures, recently developed by the Stevens Group. The second aim describes one potential novel development for nanoneedles, in the field of biosensing. This involves the selective deposition of silver nanoparticles at the nanoneedle tips, in order to take advantage of Raman-spectroscopy-signal-enhancement phenomena. These were envisioned to facilitate the detection of Raman-active molecules, as well as the investigation of cellular processes. The third aim represents another direction in broadening the horizon of the nanoneedle applications, in the field of cell transfection. This concerns the fabrication of interdigitated, planar electrodes, having nanoneedle-bearing digits. This way, a portable device was envisioned, which could exploit tip-field-enhanced electroporation, as well as tight membrane interface, for improved delivery of molecules to cells. The fourth aim describes the optimisation of the fabrication of nanowires. These were interfaced with bacteria, for prokaryotic-cell transformation purposes. This project is conceptually similar to the nanoneedle-mediated delivery of molecules that has been proven in the literature, but on a smaller scale. Bacteria cells are indeed smaller that eukaryotic ones, thus smaller nanostructures were developed to deliver cargoes into them. This thesis aims to give a contribution to the field of vertically-aligned nanostructures for nanomedicine. This field needs further insights before devices can be used in clinical settings but holds great promises for the future.Open Acces

Modular Genetic Design of Multi-domain Functional Amyloids: Insights into Self-assembly and Functional Properties

© The Royal Society of Chemistry. Engineering functional amyloids through a modular genetic strategy represents new opportunities for creating multifunctional molecular materials with tailored structures and performance. Despite important advances, how fusion modules affect the self-assembly and functional properties of amyloids remains elusive. Here, using Escherichia coli curli as a model system, we systematically studied the effect of flanking domains on the structures, assembly kinetics and functions of amyloids. The designed amyloids were composed of E. coli biofilm protein CsgA (as amyloidogenic cores) and one or two flanking domains, consisting of chitin-binding domains (CBDs) from Bacillus circulans chitinase, and/or mussel foot proteins (Mfps). Incorporation of fusion domains did not disrupt the typical β-sheet structures, but indeed affected assembly rate, morphology, and stiffness of resultant fibrils. Consequently, the CsgA-fusion fibrils, particularly those containing three domains, were much shorter than the CsgA-only fibrils. Furthermore, the stiffness of the resultant fibrils was heavily affected by the structural feature of fusion domains, with β-sheet-containing domains tending to increase the Young's modulus while random coil domains decreasing the Young's modulus. In addition, fibrils containing CBD domains showed higher chitin-binding activity compared to their CBD-free counterparts. The CBD-CsgA-Mfp3 construct exhibited significantly lower binding activity than Mfp5-CsgA-CBD due to inappropriate folding of the CBD domain in the former construct, in agreement with results based upon molecular dynamics modeling. Our study provides new insights into the assembly and functional properties of designer amyloid proteins with increasing complex domain structures and lays the foundation for the future design of functional amyloid-based structures and molecular materials