A Flexible Bivalent Approach to Comprehensively Improve the Performances of Stilbazolium Dyes as Amyloid‑β Fluorescent Probes

Exploring new ways to reconstruct the structure and function of inappropriate organic fluorophores for improving amyloid-β (Aβ) fluorescent imaging performance is desired for precise detection and early diagnosis of Alzheimer’s disease (AD). With stilbazolium dyes as examples, here, we present a multipronged approach to comprehensively improved the Aβ fluorescent imaging performance through a flexible bivalent method, where a flexible carbon chain was introduced to link two monomers to form a homodimer. Our results reveal a mechanism wherein the flexible linker creates a well-defined probe with specific orientations and distinct photophysical properties. Applying this approach in combination with theoretical simulation, the homodimers exhibited a comprehensive improvement of the Aβ fluorescent imaging performance of the dye monomers, including better photostability and higher signal-to-noise (S/N) ratio, higher “off–on” near-infrared fluorescence (NIRF) response sensitivity, higher specificity and affinity to Aβ deposits, and more reasonable lipophilicity for blood–brain barrier (BBB) penetrability. The results demonstrate that flexible homodimers offer a multipronged approach to obtaining high-performance NIRF imaging reagents for the detection of Aβ deposits both in vitro and in vivo.