Single molecule counting coupled to rapid amplification enables detection of αSynuclein aggregates in cerebrospinal fluid of PD patients

α-Synuclein aggregation is a hallmark of Parkinson's disease and a promising biomarker for early detection and assessment of disease progression. The prospect of a molecular test for Parkinson's disease is materializing with the recent developments of detection methods based on amplification of synuclein seeds (e.g. RT-QuIC or PMCA). These methods have shown promising results and identifies synucleinopathies from a variety of biofluids. However, those experiments are often very long (days) and rely on precise experimental parameters. Here we adapted single molecule counting methods for the detection of α-Synuclein aggregates in cerebro-spinal fluid (CSF). This technique is performed in solution, using the approved and well-described amyloid reagent thioflavin T and a dedicated device with minimal user input. Single molecule methods enable to probe the early events in the amplification process used in RT-QuIC and a precise counting of ThT-positive aggregates. The measurements are fast (min) and show very good sensitivity, down to attograms of Synuclein aggregates when coupled with a single short, isothermal amplification step. Importantly, the use of single molecule counting also allows a refined characterization of the samples and fingerprinting of the protein aggregates present in CSF of patients. The fingerprinting of size and ThT reactivity of each individual aggregate shows a unique signature for each PD patients compared to controls and may provide new insights on synucleinopathies in the future.