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

## TL;DR

A new water-soluble dye called DANIR-2b(2OH) can detect early stages of amyloid aggregation better than the commonly used Thioflavin T, offering improved sensitivity and broader applicability.

## Contribution

Introduces DANIR-2b(2OH), a novel dye that detects early amyloid aggregates and small fibrils missed by ThT.

## Key findings

- DANIR-2b(2OH) binds to early prefibrillar aggregates and small fibrils of human Islet Amyloid Polypeptide.
- The dye provides lower noise in plate-reader assays and tracks aggregation of proteins like insulin and Aβ1–42.
- It features environment-sensitive emission and high photostability, making it suitable for real-time imaging.

## Abstract

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 Aβ1–42. 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.