Ferroptosis Revealed by a Bifunctional Nanopipette Sensing the Intracellular Iron-Reactive Oxygen under Dibenzothiophene Exposure

Ferroptosis, as an iron-dependent form of regulated cell death, is characterized by disrupted iron homeostasis and excessive lipid peroxidation. However, current methods usually rely on population analysis of a single biomarker and are unable to obtain the real-time dynamic interplay of key intracellular species. Here, we developed a bifunctional nanopipette that can synchronously determine Fe 2+ and H 2 O 2 at the single-cell level by integrating scanning electrochemical microscopy (SECM) and scanning ion conductance microscopy (SICM). This nanopipette features bifunctions, whose inner surface was modified with 1,10-phenanthroline-4-carboxylic acid (Phen-COOH) for capturing Fe 2+ detection using SICM, while the outer surface was coated with Prussian blue (PB) for catalyzing H 2 O 2 using SECM. The two interfaces were characterized by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). Their high sensitivity (Fe 2+, 0.65 nA/μM; H 2 O 2, – 0.09 nA/μM) and good selectivity are attributed to the high catalysis of PB and the capture ability of phenanthroline. In addition, the morphology of HepG2 cells was imaged in situ in real time. Ferroptosis effects on HepG2 cells under dibenzothiophene (DBT) exposure were investigated using this SECM-SICM platform. DBT exposure induced a coordinated increase in intracellular Fe 2+ and H 2 O 2, accompanied by reduced mitochondrial activity, increased malondialdehyde accumulation, and enhanced lactate dehydrogenase release, which indicate that DBT triggers ferroptosis of HepG2 cells through an iron-dependent oxidative mechanism. This work provides a single-cell analytical strategy for dissecting ferroptosis-related pathways and evaluating the toxicological effects of environmental pollutants.