Interplay between β-Chitin Nanocrystal Supramolecular Architecture and Water Structuring: Insights from Three-Dimensional Atomic force Microscopy Measurements and Molecular Dynamics Simulations

Chitin, a highly abundant natural biopolymer, holds significant promise as a renewable resource; however, the structural organization and hydration properties of its β-crystalline form remain poorly understood, which limits our understanding of its fundamental characteristics. A thorough understanding of the three-dimensional (3D) supramolecular arrangement of chitin nanocrystals (NCs) in aqueous environments is crucial for realizing their full potential. Here, we employed atomic force microscopy (AFM) in combination with molecular dynamics simulations to elucidate the surface structure of individual β-chitin NCs at the single-fiber level under varying pH conditions. In situ AFM imaging reveals a highly ordered crystalline architecture with unprecedented molecular detail and demonstrates that water intercalation expands the lattice along the <i>b</i>-axis. 3D-AFM further revealed heterogeneous, pH-dependent hydration shells, forming an intricate 3D hydrogen bonding network around β-chitin NCs, thereby establishing substantial energetic barriers to direct interactions of external ions and molecules with the chitin surface and might lead to selective biomolecular interactions. Notably, α-chitin NCs exhibit stronger hydration forces than β-chitin NCs, reflecting distinct reactivities. Understanding the molecular assembly of β-chitin chains and their interactions with water across different pH values is critical for elucidating relevant biological processes and optimizing chitin decrystallization strategies. These findings advance our mechanistic understanding of the molecular assembly of water-intercalated β-chitin NCs, thereby enabling their efficient use in renewable products and supporting the rational design of functional and sustainable bionanomaterials.