Microfluidics-Based High-Throughput Single-Cell Analysis of Reactive Oxygen Species and T Cell Exhaustion

Reactive oxygen species (ROS) play a significant role in cellular signaling and oxidative stress, with elevated levels often linked to T cell exhaustion in various pathological conditions, including cancer. However, the relationship between ROS and T cell exhaustion in acute myeloid leukemia (AML) remains unexplored. To address this, we developed a high-throughput single-cell platform─T cell exhaustion and reactive oxygen species analyzer (TEROSA). The system achieved a single-cell capture efficiency of up to 80% with a throughput of 2400 cells and enabled dynamic monitoring of triple molecules, including the intracellular mitochondrial superoxide, on-membrane T cell exhaustion marker PD-1, and secreted extracellular H₂O₂. Our study evaluated the device's performance across multiple cell lines and demonstrated its capability to assess ROS production at the single-cell level. In particular, we analyzed T cells from AML patients and found significantly elevated ROS levels and increased PD-1 expression compared to healthy donors, suggesting a potential link between ROS and T cell exhaustion in AML. These findings highlight the utility of TEROSA in advancing our understanding of T cell behavior in leukemia and underscore its potential for broader applications in single-cell analysis.