Study Rationale: The detection of protein aggregates is currently at the leading edge of diagnostic developments in Parkinson’s disease and other age-related disorders. There is demand for technologies for the detection of a small number of aggregates capable of seeding aggregation directly from clinical samples. Recently developed seed amplification assays (SAAs) allow for the sensitive detection of small quantities of pathological α-synuclein, but these assays do not allow for a quantitative and accurate analysis of seed concentration at the single aggregate level.
Hypothesis: We propose by going beyond the state-of-the-art to generate a next generation digital microfluidic toolkit for the direct quantification of single α-synuclein seeds. Through their amplification in droplets, we will gain absolute quantification of α-synuclein aggregates in patient samples.
Study Design: As digital detection approaches are based on the amplification of single aggregates per droplet, sample dilution is required, thus reducing the detection resolution. We aim to apply novel single-molecule biophysical approaches to develop a flow-based microfluidic technique for the generation of millions of droplets at a time in an automated manner. We will achieve this goal by combining confocal microscopy, direct detection and quantification of seed bearing microdroplets. In addition, we propose to compare a variety of biological samples to identify an easily accessible biospecimen that gives the most reliable and reproducible results. Finally, we will share this platform for diagnostic applications in PD patients.
Impact on Diagnosis/Treatment of Parkinson’s disease:
We expect that the proposed work will lead to two crucial advancements in the development of quantitative diagnostic SAA. First, we will generate a digital platform for early and accurate diagnosis and monitoring of PD patients. Second, we will provide a comparative assessment of biological samples for their application with this platform.
Next Steps for Development: If our project is successful, we will provide a simple to use tool kit to researchers and clinicians with the potential to be used as point-of-care in vitro diagnostics.