ANALYTICAL APPLICATIONS OF A NEW AQUEOUS PEROXYOXALATE CHEMILUMINESCENCE REAGENT. COMPARISON WITH THE TYPICAL TCPO REACTION IN ORGANIC SOLVENT

摘要

The peroxyoxalate chemiluminescent reaction has been used for the detection of fluorescent molecules in liquid chromatography and fluorophore-labeled protein and DNA bands on membranes. In this nonenzymatic reaction, an oxalate ester reacts with hydrogen peroxide and gives rise to an intermediate capable of producing the excitation of different fluorophores. Several oxalate esters have been developed but TCPO [bis(2,4,6-triclorophenyl)oxalate] is the most used. The peroxyoxalate chemiluminescent reaction is very efficient in organic media, but the low solubility and instability of TCPO in aqueous media has limited the analytical applications of this system. In this work we have examined the properties of N,N′-di(trifluoromethanesulfonyl)-N,N′-di(4-sulfobenzylmethyl)oxamide, a new water-soluble peroxyoxalate reagent developed by Barnett et al. We describe the application of this oxamide reagent to the detection of specific protein bands on polyvinylidene difluoride (PVDF) membranes.