Enhanced sensitivity of Cypridina luciferin analogue (CLA) chemiluminescence for the detection of *O2(-) with non-ionic detergents.

Superoxide anion-triggered chemiluminescence of Cypridina luciferin analogue (CLA), 2-methyl-6-phenyl-3,7-dohydroimidazo[1,2-alpha]pyrazin-3-one, is enhanced by non-ionic detergents such as Tween 20, Triton X-100 and Tween 80. At the concentration of 0.6% (v/v) the largest increase (2.7-fold) of CLA light intensity was obtained with Tween 20, followed by Tween 80 and Triton X-100. Using this detergent-amplified CLA chemiluminescence, the detection limits of xanthine and xanthine oxidase were examined at pH 7.4 and reinvestigated at pH 5.5. At pH 5.5, concentrations of xanthine and xanthine oxidase as low as 5 nmol/L and 3.85 x 10(-7) U/mL, respectively, could be accurately determined, whereas, under the experimental conditions used, at pH 7.4 the lowest concentrations of xanthine and xanthine oxidase detectable were 10 nmol/L and 3.85 x 10(-6) U/mL. The lowest detectable values of xanthine and xanthine oxidase obtained at pH 5.5 are about 400 and 10 times lower than those previously reported. The detection limit of xanthine (5 nmol/L) by this chemiluminescent-based method is about 200 and 20 times more sensitive than the determination of xanthine by enzymatic means or by HPLC with detection limits of 1 micromol/L and 0.1 micromol/L, respectively. Our data suggest that this chemiluminescent probe can detect concentrations of superoxide anion below the nanomolar range.