Analysis of Single Particles of Amyloid Beta and α‐Synuclein With Seeded Amplification for the Diagnosis of Alzheimer's and Parkinson's Disease

Neurodegenerative diseases, including Alzheimer's disease (AD) and Parkinson's disease (PD), are characterized by the pathological aggregation of specific proteins such as amyloid beta (Aβ) and α-synuclein, respectively. Early detection of these protein aggregates in biological fluids could facilitate timely diagnosis and therapeutic intervention. This study explores an aggregate amplification approach using the surface-based fluorescence intensity distribution analysis (sFIDA) method to enhance the detection sensitivity of Aβ and α-synuclein seeds. Two amplification strategies were investigated: surface-bound and solution-phase amplification. In the case of Aβ, surface-bound amplification using immobilized Aβ-specific antibodies was tested with synthetic Aβ_1-42 seeds and monomeric Aβ_1-42 as the substrate. Despite observable aggregation, self-aggregation of the monomeric substrate interfered with seed-dependent amplification, rendering the approach ineffective at physiologically relevant concentrations. Attempts to suppress self-aggregation using blocking peptides, bovine serum albumin, and truncated Aβ_11-42 substrates were unsuccessful. Solution-phase amplification followed by surface detection also failed to reliably differentiate seeded aggregation from self-aggregation, indicating that Aβ amplification is unsuitable for diagnostic applications. In contrast, α-synuclein exhibited significantly lower self-aggregation, allowing for more effective seeded amplification. Surface-bound α-synuclein amplification successfully detected synthetic seeds at nanomolar concentrations, while solution-phase amplification further improved sensitivity, enabling detection down to the picomolar range. The method was successfully applied to brain homogenates from transgenic PD model mice, demonstrating the potential for detecting α-synuclein seeds in biological samples. These findings highlight the limitations of Aβ amplification for diagnostic purposes while supporting the feasibility of α-synuclein amplification for PD detection. Future work will focus on optimizing this approach for clinical applications.