Profiling Protein Aggregate Size Using Single-Molecule Array Technology.

Protein aggregation is a central feature of many neurodegenerative diseases, yet methods to characterize aggregate size in complex biological samples remain limited. Here, we show that fluorescence intensity from individual single-molecule array (Simoa) microwells encodes size-dependent information beyond conventional digital quantification. Using defined synthetic tau assemblies, we establish that increasing aggregate size produces higher microwell brightness. Applying this technique to human brain homogenate reveals a shift toward larger tau aggregates in Alzheimer's disease compared to age-matched controls, in agreement with orthogonal measurements by single-molecule super-resolution microscopy. Brightness profiling further captures time-dependent aggregate size increase in a neuronal cell model, demonstrating sensitivity to dynamic changes in aggregation. Although resolution is limited between similarly sized small species, Simoa brightness robustly reports population-level shifts in aggregate size distributions. These findings repurpose a widely used ultrasensitive detection platform to provide high-throughput structural as well as quantitative insight into protein aggregation in biological systems.