Enzyme-triggered formation of electrochemiluminescent ruthenium complexes

A sensitive enzyme assay is described using substrates derivatized with metal-binding ligands. Enzymes catalyzed changes in the abilities of substrates to kind to the nonelectrochemiluminescent complex ruthenium(II) bis(bipyridyl), Ru(bpy)(2)(2+), to form electrochemiluminescent mixed-ligand complexes. A highly electrochemiluminescent complex was formed between Ru(bpy)(2)(2+) and picolinic acid ((1) under bar) but not picolinic acid ethyl ester ((4) under bar), permitting the detection of (4) under bar hydrolysis by pig liver esterase (PLE). Electrochemiluminescence (ECL) differences between Ru(bpy)(2)(2+) mixtures of (1) under bar and (4) under bar were detected to a lower concentration limit of 65 pM. Under the conditions used in actual enzyme assays, it was possible to detect 4.4 pM PLE and the hydrolysis of 1.3 mu M (4) under bar. in a second assay, leucine aminopeptidase (LAP) hydrolyzed 8-(L)-leucylaminoquinoline ((9) under bar) to leucine and aminoquinoline (<(10)under bar>). A mixed-ligand complex formed between ((9) under bar) and Ru(bpy)(2)(2+) was substantially more electrochemiluminescent than a complex of Ru(bpy)(2)(2+) and <(10)under bar>. ECL differences between Ru(bpy)(2)(2+) mixtures of (9) under bar and <(10)under bar> were detectable to 65 nM. Under actual enzyme assay conditions, 375 pM LAP could be detected as well as the hydrolysis of 1.3 mu M (9) under bar. (C) 1996 Academic Press, Inc.