Enhanced sensitivity of Cypridina luciferin analogue (CLA) chemiluminescence for the detection of O-.(2)- with non-ionic detergents

Superoxide anion-triggered chemiluminescence of Cypridina luciferin analogu e (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 C LA light intensity was obtained with Tween 20, followed by Tween 80 and Tri ton X-100. Using this detergent-amplified CLA chemiluminescence, the detect ion limits of xanthine and xanthine oxidase were examined at pH 7.4 and rei nvestigated at pH 5.5. At pH 5.5, concentrations of xanthine and xanthine o xidase as low as 5 nmol/L and 3.85 x 10(-7) U/mL, respectively, could be ac curately 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 xant hine and xanthine oxidase obtained at pH 5.5 are about 400 and 10 times low er than those previously reported. The detection limit. of xanthine (5 nmol /L) by this chemiluminescent-based method is about 200 and 20 times more se nsitive than the determination of xanthine by enzymatic means or by HPLC wi th detection limits of 1 mu mol/L and 0.1 mu mol/L, respectively. Our data suggest that this chemiluminescent probe can detect concentrations of super oxide anion below the nanomolar range. Copyright (C) 2001 John Wiley & Sons , Ltd.