Time-Resolved Oxygen Dynamics Reveals Redox-Selective Apoptosis Induced by Cold Atmospheric Plasma in HT-29 Colorectal Cancer Cells

Cold atmospheric plasma (CAP) has emerged as a promising anticancer approach because of its ability to selectively eliminate malignant cells. Among the proposed mechanisms of this selectivity, the Bauer theory emphasizes the synergistic action of plasma-derived hydrogen peroxide (H₂O₂) and nitrite (NO₂^-), leading to the transient generation of primary singlet oxygen (¹O₂). This early event inactivates membrane-bound catalase, allowing tumor cell-derived H₂O₂ and peroxynitrite to initiate a self-amplifying cycle that produces secondary ¹O₂, as a hallmark of CAP selectivity. To test this hypothesis, in this work, we monitored extracellular dissolved oxygen (DO) dynamics in HT-29 colorectal cancer cells treated with an argon plasma jet using time-resolved phosphorescence lifetime spectroscopy. Temporal variations in DO likely reflect the cumulative effect of rapid ¹O₂ production and its reactions with cells. A delayed surge in extracellular ¹O₂ was observed specifically in dying cancer cells within the 10-20 min window predicted by the model. Intracellular ROS imaging confirmed a strong correlation between intracellular ROS, extracellular ¹O₂ dynamics, and viability loss. Together, these results provide mechanistic validation of Bauer's redox model and suggest that early oxygen dynamics after CAP exposure can serve as predictive markers for treatment efficacy in plasma or photodynamic therapies.