Understanding the dynamic molecular activities within single live cells is essential for deciphering cellular heterogeneity, differentiation, senescence, and disease progression. However, conventional single-cell analysis methods, which typically rely on micromanipulation or extraction followed by offline measurements, fail to capture in situ real-time dynamics.

a research team led by Prof. WU Hai-Chen from the Institute of Chemistry, Chinese Academy of Sciences (ICCAS), and Prof. LIU Lei from Xihua University, developed an innovative nanopore probe that enables real-time, multiplexed, and label-free monitoring of biomolecules within single living cells.

The device integrates an Al2O3 nanostraw membrane for efficient and nondestructive molecular extraction and a glass nanopore membrane embedded with a functionalized Mycobacterium smegmatis porin A protein nanopore (MspA-Phen-Cu) for single-channel electrical detection. Under the combined influence of diffusion and an applied electric field, intracellular molecules migrate through the nanostraw membrane toward the protein nanopore. Their translocation generates characteristic current signatures, allowing identification of individual molecules and quantification of their concentrations.

Using an oxygen-glucose deprivation (OGD) hippocampal neuron model to simulate ischemic-hypoxic stress, the researchers simultaneously tracked dynamic changes in three key molecules—glutamate (Glu), ascorbic acid (AA), and adenosine triphosphate (ATP)—all of which are critically involved in excitotoxic neuronal edema. The results confirmed that excessive activation of NMDA receptors (NMDARs) serves as a key regulatory switch in excitotoxic neuronal edema.

Compared with existing single-cell analysis technologies, this platform offers several notable advantages: minimal cellular disruption for long-term continuous observation, in situ extraction with real-time monitoring, and simultaneous detection of multiple target molecules.

This work represents a successful application of nanopore sensing inside living cells, offering a powerful platform for probing cellular processes and disease mechanisms at single-molecule resolution.

Nanopore probes for in situ molecular extraction and detection in single cells (Image by Xiaobin Huang)

This study was published in Proceedings of the National Academy of Sciences of the United States of America(PNAS).

Contact:

Prof. WU Hai-Chen

Institute of Chemistry, Chinese Academy of Sciences

Email: haichenwu@iccas.ac.cn