Molecular Dynamics Calculations of Thioflavin-T on Self-Assembled Peptides

Peptides have the remarkable ability to self-assemble into well-ordered structures on two-dimensional (2D) materials like graphene, making them promising candidates for biosensor applications due to their design flexibility and biocompatibility [1]. Recent studies have underscored the potential of graphene biosensors functionalized with peptides, mimicking olfactory receptors for odor-sensing applications [2,3]. Understanding the surface structure of self-assembled peptides on 2D material-based biosensors is vital for grasping their performance. Previous research [4] has shown that peptide self-assemblies form large domains exceeding 100 micrometers in diameter, yet the formation mechanism of these domains remains poorly understood. To date, atomic force microscopy (AFM) has been the primary tool for precisely observing these peptide structures with high spatial resolution. However, AFM is not suitable for large-area measurements in a short time.<br/>To address this limitation, we employ Thioflavin-T (ThT) fluorescent assays [5] to examine peptide self-assembly, which potentially allows us to obtain label-free fluorescent images with a wide field of view. We utilized peptides with dipeptide repeats of glycine and alanine, forming ordered monomolecular thick structures on 2D materials [4]. Hexagonal boron nitride (h-BN) surfaces served as substrates for peptide self-assembly and fluorescent microscopy. h-BN, an optically transparent member of 2D materials, is well-suited for fluorescent microscopy. ThT, known for its strong fluorescence when bound to β-sheet structures, is widely used in observing amyloid fibers associated with Alzheimer’s disease. Given that the peptides in this study are expected to form β-sheet structures, we investigate the feasibility of using ThT for real-time observation of the macroscopic surface self-assembly process of peptides.<br/>In our experiments, h-BN flakes were transferred onto glass slips, and peptide solution was applied to the surface. After a 1-hour incubation, AFM measurements revealed that peptides formed long-range ordered structures on h-BN. Upon introducing ThT into the solution, fluorescent microscopy detected strong fluorescence in regions with ordered peptide structures. The peptide domain size on the surface spanned tens of micrometers, surpassing the field of view of AFM measurements. The fluorescent measurement with ThT molecules enabled visualization of the macroscopic structures of peptides. Additionally, weak fluorescence was observed on the excess area of the h-BN surface, indicating the presence of peptide and/or ThT aggregates.<br/>The ThT association with self-assembled peptides was further studied using molecular dynamics simulations. Initially, four peptides were placed on an graphene surface, and calculations showed stable β-sheet-like structures in an anti-parallel form. Upon adding ThT to the system, a ThT molecule was stably immobilized on the self-assembled peptides with a β-sheet conformation, corroborating the experimental results.<br/>These findings highlight ThT's potential as a tool for assessing macroscopic peptide self-assembly over wide areas. Real-time monitoring of these processes under liquid conditions using fluorescence microscopy provides insights into the spatial uniformity of molecular thin films, critical for enhancing the activity of peptide-based bio-probes on graphene biosensors. This methodology paves the way for developing improved peptide biosensors with enhanced functionality and performance.<br/>[1] Y. Hayamizu, et al. Sci Rep 6, 33778 (2016)<br/>[2] C. Homma, et al. Biosens. Bioelectron., 2023, 224, 115047<br/>[3] T. Rungreungthanapol, et al. ACS. Anal. Chem. 2023, 95, 9, 4556-4563<br/>[4] P. Li, et al. ACS App. Mat. Inter., 2019, 11, 23, 20670-20677<br/>[5] T. N. Tikhonova, et al. Angew. Chem., Int. Ed., 2021, 60, 25339-25345