Metal ions homeostasis in mitochondria is tightly linked to cellular physiology and ischemic pathogenesis. Zn2+ and Ca2+ are two pivotal metal ions highly enriched in mitochondria, and their abnormal accumulation contributes to ischemic mitochondrial dysfunction and cell death. However, existing strategies struggle to track correlated Zn2+/Ca2+ fluctuations in living mitochondria under ischemic‑mimicking conditions, which hinders direct observation of their coupled responses in a cellular model of cerebral ischemia. Herein, we develop a dual-sensing θ-nanopipette for label-free, simultaneous, and quantitative detection of mitochondrial Zn2+ and Ca2+ at the single-cell level. The θ-nanopipette integrates two... More
Metal ions homeostasis in mitochondria is tightly linked to cellular physiology and ischemic pathogenesis. Zn2+ and Ca2+ are two pivotal metal ions highly enriched in mitochondria, and their abnormal accumulation contributes to ischemic mitochondrial dysfunction and cell death. However, existing strategies struggle to track correlated Zn2+/Ca2+ fluctuations in living mitochondria under ischemic‑mimicking conditions, which hinders direct observation of their coupled responses in a cellular model of cerebral ischemia. Herein, we develop a dual-sensing θ-nanopipette for label-free, simultaneous, and quantitative detection of mitochondrial Zn2+ and Ca2+ at the single-cell level. The θ-nanopipette integrates two independent channels: Channel Ⅰ employs Zn2+-specific aptamers and ionic current rectification (ICR) for Zn2+ sensing with a detection limit of 10 pM, while channel Ⅱ utilizes a Ca2+-selective membrane for potentiometric detection of Ca2+ down to 0.1 μM. This biosensor exhibits high selectivity, minimal invasiveness, and excellent stability for intracellular measurements. Using this platform, we reveal an approximate 8.5-fold overload of mitochondrial Zn2+ and 10.5-fold overload of Ca2+ in ischemic U87 cells. Furthermore, we achieve accurate monitoring of the ion changes during nimodipine intervention. This work provides a powerful subcellular electrochemical tool for dissecting metal ion networks in mitochondria, holding great potential for drug evaluation and disease mechanism studies.
