Simultaneous Imaging of Cu ⁺ and Cu ² ⁺ in Neural Cells Using DNAzyme Probes Reveals Mechanistic Link Between Copper Redox Imbalance and Amyloid Pathology

ABSTRACT Copper dysregulation is implicated in neurodegenerative diseases such as Alzheimer's disease (AD), yet its precise role in neuronal death remains unclear. To address this issue, here, we introduce a pair of Cu + and Cu 2 + specific DNAzymes‐based fluorescent probes, for the first time, enabling simultaneous visualization of both redox states of copper in single living neurons. Using this dual‐color system, we found that amyloid‐beta (Aβ) oligomerization promotes intracellular copper accumulation, distinct from that induced by artificial ionophore loading. Elevated Cu + drives reactive oxygen species (ROS) generation, lipoylated protein aggregation, and FDX1‐dependent cuproptosis, while Cu + chelation or FDX1 knockdown completely prevents cell death. In contrast, ROS scavengers only partially rescue viability, demonstrating that neuronal death is driven by copper overload, not oxidative stress itself. These findings redefine the mechanistic framework linking copper redox imbalance to Aβ pathology and neuronal vulnerability and demonstrate a selective, sensitive approach for monitoring copper homeostasis and its disruption in neurodegenerative disease.