Objective: Alpha-synuclein seed amplification assays (SAA) are highly sensitive and specific for detecting endogenous alpha-synuclein (Syn) seeds and are emerging as diagnostic tools for early detection of Parkinson’s disease. However, the readout remains largely qualitative. Here, we combine established SAA technology with highly sensitive single-molecule spectroscopy to detect and quantify individual Syn seeds.

Background: SAA is a promising diagnostic approach for Parkinson’s disease and other synucleinopathies by identifying misfolded Syn proteins. The assay exploits prion-like replication, a templating mechanism in which aggregated proteins catalyze the conversion of monomeric proteins into misfolded amyloid forms. Conversion, in the presence of seeds from patient tissues or biofluids, is detected by fluorescence. SAA can diagnose PD and related synucleinopathies with high sensitivity (85–90%) and specificity (>95%), including at pre-symptomatic stages. However, interpretation beyond a positive/negative result remains difficult, and quantitative versions of the assay are lacking. This limitation restricts the use of SAA for monitoring disease progression and in clinical trials.

Method: Our approach introduces a seed quantification method based on single-molecule spectroscopy, enabling fluorescence measurements with analytical sensitivity up to a million-fold higher than conventional plate readers. Measurements are performed using a compact, 3D-printed microscope that automatically counts and measures seeds. A small confocal volume is generated by focusing a laser into solution. Individual seeds are detected as fluorescence bursts as they diffuse through the focal volume. The number of events correlates directly with the absolute concentration of aggregates.

Results: We optimized protocols for single-molecule SAA (smSAA). Using recombinant fibrils, we determined a limit of detection of ~10 fM seeds and confirmed assay specificity and precision. Absolute concentrations of seeds aggregates were measured in serum and cerebrospinal fluid (CSF) prior to amplification. We successfully amplified seeds in serum and plasma and showed that smSAA discriminates between PD patients and controls.

Conclusion: These results provide proof-of-concept that smSAA can be applied to different matrices and deliver quantitative measurements of Syn seeds.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/smsaa-developing-a-quantitative-seed-amplification-assay-with-single-molecule-spectroscopy/