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

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[(trifluoromethyl-sulphonyl)imino]trimethylene-bis(4- methylmorpholinium)trifluoromethanesulphonate as a chemiluminogenic reagent and 2,4,6,8-tetramorpholinopyrimido[5,4- d]pyrimidine as fluorophore. The standard curve for hydrogen peroxide was linear over the range 1 x 10(-7)-1 x 10(-4) mol/L. Relative standard deviations for oxidase assays were 5.1-12.7% (n = 10). Detection limits were 1 x 10(-3) U/mL for uricase, 5 x 10(-4) U/mL for choline oxidase, 5 x 10(-3) U/mL for cholesterol oxidase and 5 x 10(-4) U/mL xanthine oxidase (sample to blank ratio, 3).

Determination of aromatic compounds by high‐performance liquid chromatography with on‐line photoreactor and peroxyoxalate chemiluminescence detection

“…Therefore, a mixture of 0.25 mmol/L TCPO and 0.75 μmol/L TMP were chosen for further experiments (21).…”

Analytical Studies on the Development of High-Performance Liquid Chromatographic Methods with Fluorescence or Chemiluminescence Detections and Their Practical Applications

“…37 Evaluations of the derivatives as a PO-CL reagent by FIA and batch methods were performed, and the combinations of TCPO and 2,4,6,8-tetrathiomorpholinopyrimido [5,4-d]pyrimidine (compound 47, TMP) gave more than 10 times larger RCI compared to those of representative fluorophores such as 9-aminoacridine, fluorescein, rhodamine B, 9,10-diphenylanthracene and perylene. 38,39 By using this combination, sensitive determination of hydrogen peroxide was achieved with an LOD of 10 nM at a signal-toblank ratio of 3. 40 Furthermore, substrates for oxidase such as glucose, uric acid, 41 mono-acetyl polyamines and polyamines 42 could be determined.…”

Chemiluminescent determination of hydrogen peroxide with 9-acridinecarbonylimidazole and use in measurement of glucose oxidase and alkaline phosphatase activity

“…Oxalate esters and amides have also been used to detect peroxide (6,18,19). A fluorescent compound must also be present.…”

“…In (18) the oxalate ester in ethyl acetate is added to the enzyme reaction plus fluorophore and mixed immediately before reading the chemiluminescence. In (19) both the fluorophore and the oxalate compound are added in water-miscible acetonitrile. In fact, the chemiluminescent reaction contains 50 mL enzyme reaction and 300 mL acetonitrile.…”