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 bind 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) but not picolinic acid ethyl ester (4), permitting the detection of 4 hydrolysis by pig liver esterase (PLE). Electrochemiluminescence (ECL) differences between Ru(bpy)2(2+) mixtures of 1 and 4 were detected to a lower concentration limit of 65pM. Under the conditions used in actual enzyme assays, it was possible to detect 4.4 pM PLE and the hydrolysis of 1.3 microM 4. In a second assay, leucine aminopeptidase (LAP) hydrolyzed 8-(L)-leucylaminoquinoline (9) to leucine and aminoquinoline (10). A mixed-ligand complex formed between (9) and Ru(bpy)2 (2+) was substantially more electrochemiluminescent than a complex of Ru(bpy)2(2+) and 10. ECL differences between Ru(bpy)2(2+) mixtures of 9 and 10 were detectable to 65 nM. Under actual enzyme assay conditions, 375 pM LAP could be detected as well as the hydrolysis of 1.3 microM 9.
Dong
L
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IGEN, Inc., Gaithersburg, Maryland 20877, USA.
Martin
M T
MT
D015082:2,2'-Dipyridyl / Q000031:analogs & derivatives* / Q000737:chemistry; D002384:Catalysis; D004563:Electrochemistry; D004950:Esterases; D006868:Hydrolysis; D007931:Leucyl Aminopeptidase; D008163:Luminescent Measurements; D015394:Molecular Structure; D009942:Organometallic Compounds / Q000737:chemistry*; D010848:Picolinic Acids / Q000737:chemistry; D012680:Sensitivity and Specificity
D009942:Organometallic Compounds; D010848:Picolinic Acids; C103599:bis(bipyridyl)ruthenium(II); D015082:2,2'-Dipyridyl; D004950:Esterases; D007931:Leucyl Aminopeptidase; C030614:picolinic acid