Nanoparticle size-dependent structural observability revealed by surface-enhanced Raman scattering in Alzheimer's...

Understanding the structural heterogeneity of protein aggregation in complex biological environments remains a central challenge in biophysical chemistry. Transient and partially ordered aggregation intermediates are particularly difficult to characterize because they exhibit structural diversity, low abundance, and weak spectroscopic signatures. The present within-subject repeated-measures study investigated how gold nanoparticle (AuNP) size influences plasmon-enhanced vibrational spectroscopy used to probe amyloid aggregation in situ. Hippocampal tissue sections from an Alzheimer's disease (AD) rat model were examined using AuNPs of two distinct diameters (20 nm and 80 nm). Surface-enhanced Raman scattering (SERS) spectroscopy combined with three-dimensional (3D) Raman imaging was used to investigate nanoparticle aggregation behavior, spectral enhancement, and structural observability within a heterogeneous biological matrix. The present study builds upon our previous observations that Aβ-coated 20 nm AuNPs exhibit nanoparticle-size-dependent quasi-reversible aggregation behavior, suggesting that nanoparticle size influences the structural states accessible during protein assembly. Accordingly, the primary objective of the present work was to clarify how nanoparticle size governs structural observability and molecular selectivity during in situ SERS analysis of amyloid aggregation. The results reveal a pronounced nanoparticle-size dependence in the SERS response. Smaller nanoparticles preferentially enhance vibrational features associated with dynamic and weakly ordered protein conformations characteristic of aggregation intermediates. The preferential observation of several localized vibrational modes in the 20 nm AuNP datasets further indicates that the reduced spectral occupancy should not be interpreted as reduced analytical sensitivity. Rather, it implies selective access to conformationally diverse and dynamically evolving molecular environments that are not strongly represented in the 80 nm datasets, consistent with previously reported nanoparticle-size-dependent quasi-reversible aggregation behavior. In contrast, 80 nm nanoparticles preferentially enhance vibrational features associated with more extended and highly ordered aggregate structures. Three-dimensional Raman imaging further demonstrated that these distinct structural states are associated with markedly different aggregate architectures and spatial extents within hippocampal tissue from the AD rat model, consistent with nanoparticle-size-dependent structural observability. These findings establish nanoparticle size as a key physical parameter governing plasmon-molecule coupling and structural observability in SERS. The use of multiple nanoparticle sizes therefore provides a practical strategy for resolving weakly ordered aggregation-associated states in situ and establishes a spectroscopic framework for investigating protein assembly pathways in complex biological environments.