Long-term amyloid aggregation kinetic assay and rapid amyloid clustering enabled by programmable microdroplet water extraction

Amyloids are misfolded protein aggregates linked to neurodegenerative diseases. Their aggregation kinetics are influenced by numerous variables and exhibit autocatalytic and stochastic behavior, prompting the use of microdroplet-based assays to enhance statistical robustness. However, uncontrolled water extraction from microdroplets during incubation alters solute concentration, limits accessible temperature and reaction time ranges, and limits experimental versatility. Here, we introduce a microdroplet handling strategy that enables programmable microdroplet water extraction. Suppressing water extraction enables long-term aggregation kinetic measurements for up to 30 days at 60°C, while promoting water extraction induces amyloid clustering within 1 hour. Using this technique, we investigated aggregation kinetics as a function of droplet size, protein concentration, and incubation time and conducted preliminary studies using microdroplet-derived amyloid clusters (MDACs). These advancements expand the experimental flexibility of droplet-based assays and provide a reproducible in vitro platform for amyloid aggregation and clustering, with potential applications in downstream screening and biological studies.