Fluorescence Imaging of Self-Assembled Peptides on h-BN Using Thioflavin-T

Peptides possess the capability to self-assemble into well-ordered structures on two-dimensional (2D) materials, such as graphene, rendering them promising candidates for biosensor applications owing to their design flexibility and biocompatibility [1]. Recent research has highlighted the potential of graphene biosensors functionalized with peptides, emulating olfactory receptors for applications in odor sensing [2,3]. Investigating the surface structure of self-assembled peptides on 2D material-based biosensors is crucial for comprehending their performance. Atomic force microscopy (AFM) with high spatial resolution has conventionally been employed for the precise observation of these peptide structures [4].<br/>Nevertheless, due to its limited field of view, AFM is unsuitable for the in-situ observation of macroscopic structures of self-assembled peptides with sizes ranging in the hundreds of micrometers. This study focuses on the in-situ observation of macroscopic structures of self-assembled peptides on hexagonal boron nitride (h-BN) surfaces using fluorescence imaging with Thioflavin-T (ThT) as a fluorescent marker. h-BN, transparent in the visible region, is well-suited for fluorescent microscopy as a transparent substrate. ThT is known to exhibit strong fluorescence when attached to β-sheet structures, commonly found in amyloid fibers associated with diseases like Alzheimer's. Given that the peptides in this study are expected to form β-sheet structures, we assessed the feasibility of using ThT for real-time observation of the macroscopic surface self-assembly process [5].<br/>AFM measurements unveiled that peptides formed long-range ordered structures on h-BN. Subsequently, under fluorescent microscopy, we observed strong fluorescence in regions where the ordered peptide structures existed. The size of the domain of ordered peptides on the surface spanned over a few tens of micrometers in length. This size exceeds the field of view of AFM measurements, and the fluorescent measurement with ThT molecules enables us to visualize the macroscopic self-assembled structures of peptides. Furthermore, weak fluorescence was also detected on partial surfaces of h-BN containing amorphous peptides. These findings underscore the potential of ThT as a tool for assessing the macroscopic self-assembly of peptides over a wide area. The ability to monitor these processes in real-time under liquid conditions using fluorescence microscopy provides insights into the spatial uniformity of molecular thin films, a critical aspect for enhancing the activity of peptide-based bio-probes on graphene biosensors. This methodology opens avenues for the development of 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