Click-Chemistry-Enabled Nanopipettes for the Capture and Dynamic Analysis of a Single Mitochondrion inside One Living Cell

Abstract

The in-depth study of single cells requires the dynamically molecular information in one particular nanometer-sized organelle in a living cell, which is difficult to achieve using current methods. Due to high efficiency of click chemistry, a new nanoelectrode-based pipette architecture with dibenzocyclooctyne at the tip is designed to realize fast conjugation with azide group-containing triphenylphosphine, which targets mitochondrial membranes. The covalent binding of one mitochondrion at the tip of the nanopipette allows a small region of the membrane to be isolated on the Pt surface inside the nanopipette. Therefore, the release of reactive oxygen species (ROS) from the mitochondrion is monitored, which is not interfered by the species present in the cytosol. The dynamic tracking of ROS release from one mitochondrion reveals the distinctive "ROS-induced ROS release" within the mitochondria. Further study of RSL3-induced ferroptosis using nanopipettes provides direct evidence for supporting the noninvolvement of glutathione peroxidase 4 in the mitochondria during RSL3-induced ROS generation, which has not previously been observed at the single-mitochondrion level. Eventually, this established strategy should overcome the existing challenge of the dynamic measurement of one special organelle in the complicated intracellular environment, which opens a new direction for electroanalysis in subcellular analysis.