Abstract

The regulation of reactive oxygen species (ROS) such as superoxide (SO) and nitric oxide (NO) is crucial in biology, influencing metabolism and signaling pathways. Imbalances in these species lead to oxidative stress and various diseases. Traditional methods for measuring SO and NO face challenges in terms of sensitivity and specificity, particularly in complex biological matrices. This report introduces bioluminescent probes that leverage the intrinsic sensitivity of bioluminescence for direct and selective detection of SO and NO. These probes release analogs of d-luciferin upon reaction with their target ROS. Following addition of luciferase, luminescence is generated proportional to the amount of accumulated luciferin, allowing for quantitation of SO or NO. Both probes exhibit high specificity, confirmed through cell-free assays and cell-based studies in macrophages, demonstrating their utility in measuring cellular SO and NO production. These assays offer a robust, high-throughput platform for studying ROS, providing direct insights into oxidative stress-related mechanisms.

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