A chemiluminescence fiber-optic biosensor system for the determination of glutamine in mammalian cell cultures.
A chemiluminescence fiber-optic biosensor system has been developed for determining glutamine in hybridoma cell cultures producing monoclonal antibodies against viral surface antigens. Glutaminase and glutamate oxidase (GLO) were immobilized onto aminopropyl glass beads via glutaraldehyde activation separately and packed in a column. Two separate columns containing immobilized GLO and catalase were placed upstream to eliminate endogenous glutamate. In the presence of ferricyanide, luminol reacted with hydrogen peroxide released from the enzymatic reactions to produce a chemiluminescence (CL) light signal which was detected and quantitated with a fiber-optic system. In combination with flow injection analysis it was possible to process samples virtually identically, thus avoiding difficulties in reproducing the CL signal. There was an excellent linear relationship between the CL response and standard glutamine concentration in the range 10(-6) to 10(-3) M. A complete analysis could be performed in 2 min including sampling and washing. Each immobilized enzyme column was stable for at least 300 repeated analyses without any loss of activity. When the biosensor system was used for the determination of glutamine in spent mammalian cell cultures, the values obtained compared well with those of high-performance liquid chromatography, thus validating the applicability of the CL fiber-optic system.
Biotechnology Research Institute, National Research Council Canada, Montreal, Quebec.
M V
MV
Cattaneo
K B
KB
Male
J H
JH
Luong
D000818:Animals; D015374:Biosensing Techniques; D002478:Cells, Cultured; D004795:Enzyme Stability; D004800:Enzymes, Immobilized; D005292:Ferricyanides; D005336:Fiber Optic Technology; D005971:Glutamates / Q000032:analysis; D018698:Glutamic Acid; D005973:Glutamine / Q000032:analysis*; D006861:Hydrogen Peroxide; D008163:Luminescent Measurements; D008165:Luminol; D008322:Mammals / Q000378:metabolism*; D055100:Optical Fibers
D004800:Enzymes, Immobilized; D005292:Ferricyanides; D005971:Glutamates; D005973:Glutamine; C007931:hexacyanoferrate III; D018698:Glutamic Acid; D008165:Luminol; D006861:Hydrogen Peroxide