GLUCOSE QUANTITATION USING AN IMMOBILIZED GLUCOSE-DEHYDROGENASE ENZYME REACTOR AND A TRIS(2,2'-BIPYRIDYL) RUTHENIUM(II) CHEMILUMINESCENT SENSOR

A flow-injection analysis detection method for glucose is presented which is based on oxidation of glucose by glucose dehydrogenase with concomitant conversion of NAD+ to NADH followed by chemiluminescent detection of NADH. The glucose dehydrogenase is immobilized via glutaraldehyde crosslinking to controlled pore glass to form an immobilized enzyme reactor. The chemiluminescent reagent, tris(2,2'-bipyridyl)ruthenium(II) [Ru(bpy)3(2+)] is immobilized in a Nafion film coated on a platinum electrode to form a regenerable chemiluminescent sensor. The immobilized Ru(bpy)3(2+) is oxidized to Ru(bpy)3(2+) which then reacts with NADH produced by the enzyme reactor to yield light and Ru(bpy)3(2+). Ru(bpy)3(2+) is thus recycled and made available again. Conditions for optimum enzyme reactor efficiency and chemiluminescent detection are determined and reported for pH (about 6.5), flow-rate (2 ml min-1), and NAD+ concentration (1-2.5 mM). At the optimum conditions a Working curve is constructed where the upper limit for glucose detection is dependant on NAD+ concentration and lower detection limit is 10 muM glucose. Signal reproducibility is 1-2% relative standard deviation. The method is very selective for glucose; some interference is seen from uric acid, ascorbic acid and catechol as well as species (such as oxalate and aliphatic amines) already known to chemiluminesce with the Ru(bpy)3(2+) sensor.