Peroxyoxalate chemiluminescent assay for oxidase activities based on detecting enzymatically formed hydrogen peroxide

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Abstract
A sensitive peroxyoxalate chemiluminescent (PO‐CL) assay for activities of oxidases (uricase, choline oxidase, cholesterol oxidase and xanthine oxidase) which catalyse a formation of hydrogen peroxide was developed using 4,4′‐oxalyl‐bis[(trifluoromethylsulphonyl)imino]trimethylene‐bis(4‐methylmorpholinium)trifluoromethanesulphonate as a chemiluminogenic reagent and 2,4,6,8‐tetramorpholinopyrimido[5,4‐d]pyrimidine as a fluorophore. The standard curve for hydrogen peroxide was linear over the range 1 × 10−7‐1 × 10−4 mol/L. Relative standard deviations for oxidase assays were 5.1–12.7% (n = 10). Detection limits were 1 × 10−3 U/mL for uricase, 5 × 10−4 U/mL for choline oxidase, 5 × 10−3 U/mL for cholesterol oxidase and 5 × 10−4 U/mL xanthine oxidase (sample to blank ratio, 3).

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