On the use of L-012, a luminol-based chemiluminescent probe, for detecting superoxide and identifying inhibitors of NADPH oxidase: a reevaluation

Free Radic Biol Med. 2013 Dec;65:1310-1314. doi: 10.1016/j.freeradbiomed.2013.09.017. Epub 2013 Sep 27.


L-012, a luminol-based chemiluminescent (CL) probe, is widely used in vitro and in vivo to detect NADPH oxidase (Nox)-derived superoxide (O2(*-)) and identify Nox inhibitors. Yet understanding of the free radical chemistry of the L-012 probe is still lacking. We report that peroxidase and H2O2 induce superoxide dismutase (SOD)-sensitive, L-012-derived CL in the presence of oxygen. O2(*-) alone does not react with L-012 to emit luminescence. Self-generated O2(*-) during oxidation of L-012 and luminol analogs artifactually induce CL inhibitable by SOD. These aspects make assays based on luminol analogs less than ideal for specific detection and identification of O2(*-) and NOX inhibitors.

Keywords: 8-amino-5-chloro-7-phenylpyrido[3,4–d]pyridazine-1,4(2H,3H)dione; CAT; CL; Free radicals; HE; HRP; HX; L-012; Luminescence; Luminol; NADPH oxidase; NADPH oxidases; Nox; O(2)(−); ROS; Redox cycling; Redox probe; SOD; Superoxide radical anion; XO; catalase; chemiluminescence; diethylenetriaminepentaacetate; dtpa; horseradish peroxidase; hydroethidine; hypoxanthine; reactive oxygen species; superoxide dismutase; superoxide radical anion; xanthine oxidase.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Catalase / chemistry
  • Free Radicals
  • Hydrogen Peroxide / chemistry
  • Luminescence
  • Luminescent Measurements / methods*
  • Luminol / analogs & derivatives*
  • Luminol / chemistry
  • NADPH Oxidases / analysis*
  • NADPH Oxidases / antagonists & inhibitors
  • NADPH Oxidases / metabolism
  • Oxidation-Reduction
  • Oxygen / chemistry
  • Peroxidase / chemistry
  • Superoxide Dismutase / chemistry*
  • Superoxides / analysis*
  • Superoxides / chemistry


  • Free Radicals
  • Superoxides
  • L 012
  • Luminol
  • Hydrogen Peroxide
  • Catalase
  • Peroxidase
  • Superoxide Dismutase
  • NADPH Oxidases
  • Oxygen