Determination of fluorophor-labeled compounds based on peroxyoxalate chemiluminescence

Fluorophor-labeled compounds are determined by adding an aromatic oxalate ester and hydrogen peroxide and measuring the resulting chemiluminescence (CL). Rhodamine B is excited more efficiently than 2',7'-dichlorofluorescein and dimethylaminonapthalenesulfonic acid. Hydrogen peroxide concentration is the most important variable influencing intensity-time characteristics of the reaction. Measurements are possible in a predominantly aqueous reaction medium but precision is poor, presumably due to oxalate ester insolubility on a microscale. Increasing the percentage of organic solvent leads to improved precision and a larger signal. The detection limit for rhodamine B is around 10(-9) M for a medium containing 50% organic solvent. It is limited by variability in background CL which is associated with impurities in the solvents used for the measurement. The CL excitation efficiency decreases when rhodamine is bound to protein but increases when rhodamine is coupled to folic acid. The addition of albumin to rhodamine-labeled antialbumin causes a 30% decrease in CL intensity.