A Highly Sensitive Water-Soluble Donor-Acceptor Dye for Early-Stage Amyloid Aggregation Kinetics

Protein aggregation into amyloid fibrils underlies numerous human diseases, yet the most widely used fluorescent probe, Thioflavin T (ThT), offers an incomplete picture of the process and fails to detect certain fibril structures. Here, we introduce and characterize the photophysical properties of DANIR-2b(2OH), a water-soluble push-pull dye that overcomes these limitations. It successfully binds early prefibrillar aggregates and small fibrils of the human Islet Amyloid Polypeptide that elude detection by ThT, which we confirm by time-resolved cryo-electron microscopy of aliquots taken during the kinetic assays. We further demonstrate that DANIR-2b(2OH) can also track the aggregation of other amyloid proteins, such as insulin and Aβ_1-42. The protein-dye interaction was characterized via steady-state and time-resolved fluorescent spectroscopy. DANIR-2b(2OH) features environment-sensitive emission, high photostability, and a straightforward synthesis. Critically, it provides a substantially lower noise level in standard plate-reader assays, allowing the tracking of aggregation processes that are not visible in standard ThT measurements. This establishes DANIR-2b(2OH) as a highly sensitive and broadly applicable probe for real-time amyloid aggregation measurements and imaging.