Seed amplification assays (SAAs) are a promising avenue for the early diagnosis of neurodegenerative diseases. However, when amplifying fibrils from patient‐derived samples in multiwell plates, it is currently highly challenging to accurately quantify the aggregates. It is therefore desirable to transfer such assays into a digital format in microemulsion droplets to enable direct quantification of aggregate numbers. To achieve transfer from conventional plate‐based to the microfluidic digital format, effective seed amplification needs to be achieved inside the microdroplets. Therefore, we establish a new set of assay conditions that enable highly efficient seed amplification in plates without any shaking. However, the same set of conditions displayed a very different behavior upon transfer to a microfluidic platform where no amplification was observed. We demonstrate that this is caused by the suppression of all secondary processes that could amplify the seeds in the complete absence of mechanical perturbations inside the microdroplets. We further show that the amplification inside droplets can be achieved by subjecting the microemulsions to high‐frequency vibrations using a piezo device. Taken together, our results provide novel insights into the physical requirements of alpha‐synuclein seed amplification and demonstrate a pathway towards the development of effective digital SAAs.

The amplification of alpha‐synuclein amyloid fibrils is suppressed under fully quiescent conditions