Chemiluminescence biosensors using tris(2,2'-bipyridyl)ruthenium(II) and dehydrogenases immobilized in cation exchange polymers

Tris(2,2'-bipyridyl)ruthenium(II) (Ru(bpy)(3)(2+)) and dehydrogenase enzymes are immobilized in cation exchange polymer films to create electrogenerated chemiluminescence (ECL) biosensors. Eastman AQ and Nafion polymers were used as immobilization support in a regenerable ECL sensor for NADK and NADPH and in biosensors for glucose, lactate and ethanol. The biosensor design places an enzyme-loaded polymer film adjacent to a Ru(bpy)(3)(2+)- loaded polymer film covering a platinum electrode. The modified electrode was used in a flow injection analysis system as an ECL detector. The CL response to samples containing enzyme substrate and cofactor (NAD(+)) result from the Ru(bpy)(3)(3+) ECL reaction with NADH produced by the enzyme. The analytical response to NADH using an AQ film modified with Ru(bpy)(3)(2+) was similar to that seen for Nafion films modified with Ru(bpy)(3)(2+). The coimmobilization of Ru(bpy)(3)(2+) and dehydrogenase enzymes in the same film showed reduced enzyme activity which led to various biosensor designs with adjacent layers. The ECL biosensor concept was shown applicable for both NAD(+)-and NADP(+)-dependent enzymes, where NADPH detection ranged from 0.01 to 100 mu M NADPH. (C) 1997 Published by Elsevier Science Limited.