Alkyne-tagged SERS nanoprobe for understanding Cu⁺ and Cu²⁺ conversion in cuproptosis processes

Simultaneously quantifying mitochondrial Cu⁺ and Cu²⁺ levels is crucial for evaluating the molecular mechanisms of copper accumulation-involved pathological processes. Here, a series of molecules containing various diacetylene derivatives as Raman reporters are designed and synthesized, and the alkyne-tagged SERS probe is created for determination Cu⁺ and Cu²⁺ with high selectivity and sensitivity. The developed SERS probe generates well-separated distinguishable Raman fingerprint peaks with built-in corrections in the cellular silent region, resulting in accurate quantification of Cu⁺ and Cu²⁺. The present probe demonstrates high tempo-spatial resolution for real-time imaging and simultaneously quantifying mitochondrial Cu⁺ and Cu²⁺ with long-term stability benefiting from the probe assembly with designed Au-C≡C groups. Using this powerful tool, it is found that mitochondrial Cu⁺ and Cu²⁺ increase during ischemia are associated with breakdown of proteins containing copper as well as conversion of Cu⁺ and Cu²⁺. Meanwhile, we observe that parts of Cu⁺ and Cu²⁺ are transported out of neurons by ATPase. More importantly, cuproptosis in neurons is found including the oxidative stress process caused by the conversion of Cu⁺ to Cu²⁺, which dominates at the early stage (<9 h), and subsequent proteotoxic stress. Both oxidative and proteotoxic stresses contribute to neuronal death.