In situ and real-time monitoring of intracellular activities in single live cells using a nanopore probe.

Monitoring molecular activities within single live cells is vital for understanding cellular differentiation, senescence, heterogeneity, and disease progression. However, conventional single-cell analyses often rely on micromanipulation or extraction followed by downstream measurements, which cannot capture in situ real-time dynamics. Fluorescent labeling and electrochemical methods provide temporal resolution but face limitations in labeling, substrate scope, and multiplexing. Here, we present a nanopore probe that enables real-time, multiplexed monitoring of intracellular activities in single live cells. The device integrates an aluminum oxide nanostraw membrane for molecular extraction and a glass nanopore membrane for single-channel electrical detection. Using a hippocampal neuron model of ischemia-hypoxia, we simultaneously tracked dynamic changes in intracellular glutamate, ascorbic acid, and adenosine triphosphate-three key molecules involved in oxygen-glucose deprivation-induced neuronal edema. Our findings establish this nanopore probe as a powerful platform for real-time, label-free molecular profiling at the single-cell level, opening opportunities for studying disease mechanisms and therapeutic responses.