Real-Time Monitoring of Self-Aggregation of β‑Amyloid by a Fluorescent Probe Based on Ruthenium Complex

• Published in: Analytical chemistry (Washington) 2020-02, Vol.92 (4), p.2953-2960
• Main Authors: Yu, Hui-juan, Zhao, Wei, Xie, Mengting, Li, Xiaoqing, Sun, Ming, He, Jun, Wang, Lu, Yu, Lin
• Format: Article
• Language: English
• Subjects: Agglomeration; Alzheimer's disease; Amyloid beta-Peptides - chemistry; Animals; Atomic force microscopy; Brain; Chemistry; Confocal microscopy; Coordination Complexes - chemical synthesis; Coordination Complexes - chemistry; Electron microscopy; Fibrils; Fluorescence; Fluorescence microscopy; Fluorescent Dyes - chemical synthesis; Fluorescent Dyes - chemistry; Fluorescent indicators; In vitro methods and tests; In vivo methods and tests; Indoles; Microscopy; Molecular Structure; Monitoring; Monomers; Morphology; Neurodegenerative diseases; Neurotoxicity; NMR; Nuclear magnetic resonance; Nucleation; Oligomerization; Oligomers; Pathogenesis; PC12 Cells; Perturbation; Protein Aggregates; Rats; Real time; Ruthenium; Ruthenium - chemistry; Ruthenium compounds; Surface Plasmon Resonance; Therapeutic applications; Time Factors; β-Amyloid
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/acs.analchem.9b03566

Self-accumulation of amyloid-β protein (Aβ) into insoluble fibrils is the major hallmark of Alzheimer’s disease. Real-time monitoring of fibril growth is essential for clarifying the mechanism underlying aggregation and discovering therapeutic targets. A variety of approaches including NMR, electron microscopy (EM), atomic force microscopy (AFM), and total internal reflection fluorescence microscopy (TIRFM) have been explored to monitor the fibril growth or reveal morphology of Aβ aggregates. However, none of the methods allow real-time observation under physiological conditions while without any perturbations. Here, we present a fluorescent probe [Ru­(phen)2(fipc)]2+ (Ru-fipc) (phen = 1,10-phenanthroline, fipc = 5-fluoro-N-(1,10-phenanthrolin-5-yl)-1H-indole-2-carboxamide) that can bind to all the Aβ forms, i.e., monomers, oligomers, and fibrils, while not perturbing aggregation. Using this probe in combination with laser confocal microscopy, the entire aggregation process could be clearly and exactly imaged at the single fibril level. The reliability of Ru-fipc was confirmed based on colocalization with thioflavin T (ThT). Importantly, Ru-fipc can be used to monitor the very early nucleation and oligomerization process, which is thought to be a critical step in the development of neurotoxicity while it cannot be visualized with ThT. To our knowledge, this is the first fluorescent probe developed for real-time monitoring of Aβ aggregation, especially for the very early assembly stage, in solution with minimal perturbation. This method is suitable for in vitro and in vivo studies. We believe this would provide a valuable complementary tool for the study of pathogenesis and discovery of therapeutic targets of neurodegenerative diseases.