A Dual-Functional Two-Photon PLIM Probe Based on Ruthenium(II) Complex: Sequence Selectivity and Aβ Aggregation...

A key pathological feature of Alzheimer's disease (AD) is β-amyloid (Aβ) plaque formation from abnormal peptide aggregation. In addition to full-length Aβ_1-42, various N/C-truncated Aβ fragments exist in the human brain and are found to be associated with AD etiology. However, the precise toxic Aβ species, aggregation states, and neurotoxic mechanisms remain unclear, highlighting the urgent need for probes with sequence selectivity and real-time aggregation monitoring capability─functions lacking in reported Aβ probes. Herein, we present Ru-dmpip, a two-photon phosphorescence lifetime imaging microscopy (PLIM) probe synthesized by conjugating a hydrophilic ruthenium complex and a hydrophobic ThT moiety. Ru-dmpip exhibits high specificity for Aβ_X-42 peptides (Aβ_1-42/Aβ_11-42), with 7.4/3.7-fold higher affinity than Aβ_1-40, and can bind all Aβ_X-42 aggregated states (monomers/oligomers/fibrils), inducing sensitive phosphorescence intensity and lifetime changes (the lifetime increased from 436 to approximately 900 ns). It can visualize Aβ_1-42 aggregation at the single-fiber level, enable in vitro cellular imaging, and specifically identify plaques formed by Aβ_1-42 in APP-PS1 transgenic mouse brain sections. Its ultralong excited-state lifetime can effectively mitigate interference from background, allowing clear observation of loose small oligomers surrounding plaques─species that are barely detectable via conventional spectral imaging. Binding mechanism studies show Ru-dmpip interacts with Aβ_1-42 via hydrogen bonding with the C-terminus and electrostatic attraction with the N-terminus. As the first two-photon PLIM probe integrating sequence selectivity and aggregation monitoring, Ru-dmpip provides a valuable tool for AD pathological research and inspires the design of sequence-specific Aβ sensors.