Detecting alpha-synuclein aggregates with small molecules on single-molecule array

Protein aggregates are promising biomarkers for early diagnosis of neurodegenerative disorders. Single-Molecule Array (SiMoA) is a powerful method to detect these aggregates at ultra-low concentrations in biofluids. Herein, we report a next-generation SiMoA assay using chemically synthesized small molecules, rather than antibodies, to capture alpha-synuclein aggregates, a protein hallmark in Parkinson's Disease and other synucleinopathies. These small molecule-based capturing agents contain aggregate-binding head groups, and a backbone functionalized with a primary amine for bead conjugation in the SiMoA assay. The most promising molecule, BF-79-2, captured recombinant alpha-synuclein aggregates, specifically excluding monomers, at picomolar concentrations. BF-79-2 also captured alpha-synuclein aggregates in human blood. Replacing antibodies with small molecules as capturing agents on the SiMoA platform enhances the assay versatility, since small molecules can be screened <i>in silico</i> and synthesized without laborious molecular biology techniques. The application of small molecules as capturing agents broadens the capabilities of the SiMoA platform, rendering it more adaptable for biomarker discovery and disease diagnostics.