Ma. Motsenbocker et K. Kondo, IMPROVEMENTS TO ENHANCED HORSERADISH-PEROXIDASE DETECTION SENSITIVITY, Journal of bioluminescence and chemiluminescence, 9(1), 1994, pp. 15-20
At very low horseradish peroxidase (HRP) concentrations, the enhanced
chemiluminescence reaction is often characterized by a lag time betwee
n initiation of the reaction and beginning of light output. In this st
udy, four treatments of luminol solution were examined in an effort to
remove the lag time and to improve chemiluminescence light output. Ad
dition of ammonium persulphate stimulated light output more than tenfo
ld. Ultraviolet irradiation and photoactive dye pretreatment of lumino
l solution both increased light output fourfold. Luminol purity was th
e most important factor affecting detection sensitivity. Recrystalliza
tion of luminol from base improved the detection limit 13-fold althoug
h there was an improvement in the detection limit from 13 attomoles pe
r millilitre to 5 attomoles per millilitre with highly purified lumino
l when photoactive dye pretreatment was utilized. The results are cons
istent with a simple interference mechanism whereby enhancer radicals
produced by the enzyme are preferentially quenched by contaminants pre
sent in the luminol, in the enhancer and in the solvent used to dissol
ve the enhancer. Consumption of these interferences prior to light emi
ssion results in a lag time and a less favourable HRP detection limit.