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 glucise 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) 2+ 3] is immobilized in a Nafion film coated on a platinum electrode to form a regenerable chemiluminescent sensor. The immobilized Ru(bpy) 2+ 3 is oxidized to Ru(bpy) 3+ 3 which then reacts with NADH produced by the enzyme reactor to yield light and Ru(bpy) 2+ 3. Ru(bpy) 2+ 3 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 μM glucose. Signal reproductibility 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) 2+ 3 sensor.