Bioluminescence Resonance Energy Transfer Sensor with Tunable Conjugation Efficiency for Highly Sensitive Detection of Superoxide Anion in Tumors.

Superoxide anion (O₂^•-) is a highly reactive oxygen species (ROS) within tumor cells, and its abnormal concentrations can lead to various diseases such as cancer, inflammation, and premature aging disorders. Here, we obtained a series of bioluminescence resonance energy transfer (BRET) systems that can be used for sensitive and specific detection of O₂^•- by varying the type and reaction time of quantum dots (QDs) and combining them with different concentrations of recombinant aequorin. Among them, the recombinant aequorin-conjugated CdTe/CdSe QDs had the highest conjugation efficiency as the Aeq-QD BRET sensor, which has a remarkable energy transfer efficiency of 35.6% and an extremely low limit of 4 nM for detecting O₂^•-, which exceeds traditional chemiluminescence detection relying on coelenterazine. The applicability of this sensor for assessing tumor oxidative stress levels was also validated in diverse cell types and cancer mice. This study effectively contributes to the field of cancer research and tumor oxidative stress biology based on O₂^•- sensing.