Discriminating Monomers and Fibrils of Amyloid‐ β _1–42 With Site‐Ordered Peptide‐QMFluors

ABSTRACT

The detection of soluble amyloid‐ β (A β ), 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 A β _1–42 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.