Multiphase Silk Assembly for Two-Dimensional Bio-Crystal Film

Early insights into native silk fibroin (SF) architecture suggested that its unique structures and properties are determined by its multiscale assembly and the evolution of its secondary structure. Yet the pathways of assembly and the relationship to that evolution are poorly understood. Here we investigate SF self-assembly using in situ AFM and liquid phase infrared nanospectroscopy (nano-FTIR) and molecular dynamics. To do so, we assemble the silk at the interface between water and highly ordered pyrolytic graphite (HOPG). We find that SF grows heteroepitaxially on HOPG into highly ordered, monolayer-thick 2D nanocrystals consisting of 1D lamellae that exhibit β-sheet secondary structure and lie along the armchair direction of HOPG. Molecular dynamics simulations show that the armchair orientation is indeed energetically favored, as is polar packing to form a bilayer. As the SF concentration increases, SF assembles into multi-layers via two pathways that can occur concomitantly. One is a non-classical pathway by which a disordered metastable film forms on top of the lamellae of the first monolayer and gradually converts into the lamellar structure. The second is a classical layer-by-layer pathway by which new lamellae grow homoepitaxially on the underlying 2D lamellae nanocrystals without any evidence of an intermediate state. Appling synchrotron based tip-enhanced nano-IR to SF assembly for the first time, we demonstrate that the β-sheet conformation is adopted from largely unstructured SF in solution as the lamellae advance along the classical pathway or during the process of film transformation along the non-classical pathway. These new findings fill in the missing pieces of the puzzle showing how SF structure evolves at the liquid-solid interface and provides inspiration for the design of heterogeneous 2D SF bio-crystal film.