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The detection of superoxide anion (O 2 ●- ) in biological tissues remains challenging. Barriers to convenient and reproducible measurements include expensive equipment, custom probes, and the need for high sensitivity and specificity. The luminol derivative, L-012, has been used to measure O 2 ●- since 1993 with mixed results and concerns over specificity. The goal of this study was to better define the conditions for use and their specificity. We found that L-012 coupled with depolymerized orthovanadate, a relatively impermeable tyrosine phosphatase inhibitor, yielded a highly sensitive approach to detect extracellular O 2 ●- . In O 2 ●- producing HEK-NOX5 cells, orthovanadate increased L-012 luminescence 100-fold. The combination of L-012 and orthovanadate was highly sensitive, stable, scalable, completely reversed by superoxide dismutase, and selective for O 2 ●- generating NOXes versus NOX4, which produces H 2 O 2 . Moreover, there was no signal from cells transfected with NOS3 (NO ● ) and NOS2(ONOO - ). To exclude the effects of altered tyrosine phosphorylation, O 2 ●- was detected using non-enzymatic synthesis with phenazine methosulfate and via novel coupling of L-012 with niobium oxalate, which was less active in inducing tyrosine phosphorylation. Overall, our data shows that L-012 coupled with orthovanadate or other periodic group 5 salts yields a reliable, sensitive, and specific approach to measuring extracellular O 2 ●- in biological systems.

Competing Interests: Disclosures Detailed methods and materials are available upon reasonable request. All animal experiments were conducted under Augusta University Institutional Animal Care and Use Committee (IACUC) approval and in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals.