Real-time monitoring of cellular superoxide anion release in THP-1 cells using a catalytically amplified superoxide dismutase–based microbiosensor

Reactive oxygen species (ROS) including the superoxide anion (O₂^•−) are typically studied in cell cultures using fluorescent dyes, which provide only discrete single-point measurements. These methods lack the capabilities for assessing O₂^•− kinetics and release in a quantitative manner over long monitoring times. Herein, we present the fabrication and application of an electrochemical biosensor that enables real-time continuous monitoring of O₂^•− release in cell cultures for extended periods (> 8 h) using an O₂^•− specific microelectrode. To achieve the sensitivity and selectivity requirements for cellular sensing, we developed a biohybrid system consisting of superoxide dismutase (SOD) and Ti₃C₂T_x MXenes, deposited on a gold microwire electrode (AuME) as O₂^•− specific materials with catalytic amplification through the synergistic action of the enzyme and the biomimetic MXenes-based structure. The biosensor demonstrated a sensitivity of 18.35 nA/μM with a linear range from 147 to 930 nM in a cell culture medium. To demonstrate its robustness and practicality, we applied the biosensor to monitor O₂^•− levels in human leukemia monocytic THP-1 cells upon stimulation with lipopolysaccharide (LPS). Using this strategy, we successfully monitored LPS-induced O₂^•− in THP-1 cells, as well as the quenching effect induced by the ROS scavenger N-acetyl-l-cysteine (NAC). The biosensor is generally useful for exploring the role of oxidative stress and longitudinally monitoring O₂^•− release in cell cultures, enabling studies of biochemical processes and associated oxidative stress mechanisms in cellular and other biological environments.