Bioluminescence Resonance\nEnergy Transfer Sensor with\nTunable Conjugation Efficiency for Highly Sensitive Detection of Superoxide\nAnion in Tumors

Superoxide anion (O<sub>2</sub><sup>•–</sup>) is\na highly reactive oxygen species (ROS) within tumor cells, and its\nabnormal concentrations can lead to various diseases such as cancer,\ninflammation, and premature aging disorders. Here, we obtained a series\nof bioluminescence resonance energy transfer (BRET) systems that can\nbe used for sensitive and specific detection of O<sub>2</sub><sup>•–</sup> by varying the type and reaction time of quantum\ndots (QDs) and combining them with different concentrations of recombinant\naequorin. Among them, the recombinant aequorin-conjugated CdTe/CdSe\nQDs had the highest conjugation efficiency as the Aeq-QD BRET sensor,\nwhich has a remarkable energy transfer efficiency of 35.6% and an\nextremely low limit of 4 nM for detecting O<sub>2</sub><sup>•–</sup>, which exceeds traditional chemiluminescence detection relying on\ncoelenterazine. The applicability of this sensor for assessing tumor\noxidative stress levels was also validated in diverse cell types and\ncancer mice. This study effectively contributes to the field of cancer\nresearch and tumor oxidative stress biology based on O<sub>2</sub><sup>•–</sup> sensing.