IMPROVEMENTS TO ENHANCED HORSERADISH-PEROXIDASE DETECTION SENSITIVITY

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.