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 organelles within the intricate compartmentalized systems of eukaryotic cells poses significant challenges in biomedical research. Presently, assessment of organelle functions frequently 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 cells for in situ and concurrent acquisition of multiple key characteristics of a single mitochondrion. In one channel, mitochondrial reactive oxygen species (ROS) and associated adenosine triphosphate (ATP) production are electrochemically measured with a redox activatable probe (Apt -MB), while the parallel channel facilitates the measurement of mitochondrial Delta psi m via proton responsiveness. Quantitative assessment of the relationship between ROS and Delta psi m against ATP production has been achieved, which allows in-depth analysis of mitochondrial functions. With this approach, the results clearly show evolutionary stages of the apoptosis pathway, and highlight that 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 mitochondrial functionality, and may further inspire the development micro/nanoscale tools for multiparameter, high-fidel- ity, single -organelle analysis directly within cells.