NOVEL FIA AMPEROMETRIC BIOSENSOR SYSTEM FOR THE DETERMINATION OF GLUTAMINE IN CELL-CULTURE SYSTEMS

A flow injection analysis (FIA) biosensor system has been developed for determining glutamine in insect cell and in murine hybridoma cell cultures. Glutamate oxidase and glutaminase, respectively, were covalently immobilized onto porous aminopropyl glass beads to form an immobilized enzyme column. The hydrogen peroxide produced by the enzyme reactions was detected by an amperometric electrode (platinum vs. silver/silver chloride) poised at + 0.7 V. Among several anion exchange resins tested, endogenous glutamate was completely adsorbed onto acetate anion exchange resins. Such an ion exchanger also effectively adsorbed aspartic acid, uric acid, ascorbic acid, and acetaminophen. The FIA biosensor was linear up to 1 mM glutamine, with a lower detection limit of 10 muM, and possessed good reproducibility (relative error of +/- 1.2%). Each analysis could be performed in 3.5 min including sampling and washing with a corresponding throughput of 17 h-1. The anion exchange column could be operated continuously for 12 h for 200 analyses with diluted cell culture. The immobilized enzyme column was stable for at least 500 repeated analyses without significant loss of activity. When the biosensor system was applied to glutamine measurement in insect cell and mammalian cell cultures, the results obtained compared well with those HPLC.