Discriminating Monomers and Fibrils of Amyloid-β1-42 With Site-Ordered Peptide-QMFluors.

The detection of soluble amyloid-β (Aβ), the most neurotoxic species in early Alzheimer's disease (AD), remains a major analytical challenge. Conventional probes are designed to target the β-sheet structures of mature fibrils and are thus blind to these transient, nonfibrillar species. Here, we introduce "site-ordered engineering" strategy, a peptide-conjugated QMFluors conjugate designed to selectively target Aβ1-42 monomers with high fidelity. We identified the conjugation site order on the fluorophore as the key parameter controlling this molecular topology and self-assembly behavior. This breakthrough is enabled by a hydrogen-bond-driven recognition mechanism, fundamentally departing from classical dyes that target the β-sheets of mature fibrils. This yields an unprecedented "inverted selectivity" with a 20-fold fluorescence enhancement for monomers over fibrils. Using probe N-QM-KLVFF with super-resolution microscopy, we reveal the nanoscale "core-shell" biophysical phase diagram of plaques, visualizing dense fibrillar cores surrounded by monomeric/oligomeric halos. Furthermore, the probe demonstrates a limit of detection of pg/mL level and excellent linearity, meeting the stringent requirements for trace cerebrospinal-fluid-biomarker analysis. This work provides not only a powerful tool for early diagnosis and pathological investigation, but also establishes "aggregation-regulated accessibility" as a generalizable principle for designing probes for other dynamic amyloid protein targets.