Combining Engineered Probe Library with Tiered Screening for the Rational Discovery of Alzheimer's Diagnostic Probe with an Enhanced Signal-to-Noise Ratio.

Alzheimer's disease (AD) is neuropathologically defined by the deposition of β-amyloid (Aβ) plaques, a key diagnostic biomarker. However, current fluorescence probes targeting Aβ plaques are often limited by poor blood-brain barrier (BBB) penetration and short blood circulation half-lives, resulting in insufficient signal-to-noise ratios for in vivo imaging. To address this, we developed a rationally engineered near-infrared (NIR) fluorescent molecular probe library and implemented a tiered, closed-loop screening strategy for the discovery of probes, enabling high-fidelity in situ visualization of Aβ plaques. Through this integrated approach, we identified ADFP-2, which displays a high binding affinity for Aβ aggregates and exhibits a 34-fold fluorescence enhancement upon binding. Notably, ADFP-2 demonstrates optimal BBB penetrability and a prolonged circulation time, facilitating high-contrast in vivo imaging of Aβ deposits. This study not only provides a potent molecular tool for the early diagnosis of AD but also validates a systematic and efficient framework for probe discovery and optimization through engineered libraries and tiered screening.