Interactional analysis of single mitochondrial function directly in living cell reveals the proton circuit decoupling of mitochondria at the preliminary stage of apoptosis

Analyzing single organelle within the intricate compartmentalized systems of eukaryotic cells poses significant challenges in biomedical research. Presently, assessment of the organelle functions fre-quently relies on the disjointed application of multiple techniques, resulting in constraints on the promptness and precision of data acquisition. Herein, a dual-nanopore electrochemical biosensor has been designed in this work to house detection channels directly in living cell for in situ and concurrent acquisition of multiple key characteristics of a single mitochondrion. In one channel, mitochondrial ROS and associated ATP production are electrochemically measured with a redox acti-vatable probe (Apt-MB), while the parallel channel facilitates the measurement of mitochondrial ΔΨm via proton responsiveness. Quantitative assessment of the rela-tionship between ROS and ΔΨm against ATP production have been achieved, which allows in-depth analysis of mitochondrial functions. With this approach, the results clearly show evolu-tionary stages of the apoptosis pathway, and highlight that the mitochondrial proton circuit decoupling may serve as an earlier event preceding the well-known ROS accumulation stage. Hence, this study underscores the considerable usage of this approach for comprehensive analysis of single mitochondri-al function, and may further inspire the development of micro/nanoscale tools for multi-parameter, high-fidelity, single-organelle analysis directly within cell.

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