Monitoring glutamine in mammalian cell cultures using an amperometric biosensor.
An amperometric biosensor has been developed for monitoring glutamine in the pulsed-batch cultivation of murine hybridoma cells. Glutamine oxidase was cross-linked with bovine serum albumin (BSA) via glutaraldehyde activation and deposited on a preactivated nylon membrane. Glutaminase was then immobilized on the protein layer and the resulting membrane was attached to the sensing area of a hydrogen peroxide probe (platinum vs silver/silver chloride polarized at +0.7 V). An orthogonal test was performed to optimize the activity of the membrane for glutamine with respect to the concentrations of glutamate oxidase, BSA, glutaminase and glutaraldehyde. There was an excellent linear relationship between the biosensor's response and glutamine in the range 0.1-3 mM. The determination of glutamine could be performed in 2 min and each membrane was reused for at least 300 consecutive analyses. The data obtained also agreed well with those high-performance liquid chromatography, thus validating the applicability of the biosensor.
Biotechnology Research Institute, National Research Council Canada, Montreal, Quebec.
M V
MV
Cattaneo
J H
JH
Luong
S
S
Mercille
D000594:Amino Acid Oxidoreductases / Q000737:chemistry*; D000818:Animals; D015374:Biosensing Techniques; D002478:Cells, Cultured; D004553:Electric Conductivity; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry; D005971:Glutamates / Q000032:analysis; D018698:Glutamic Acid; D005972:Glutaminase / Q000737:chemistry*; D005973:Glutamine / Q000378:metabolism*; D006825:Hybridomas / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D004800:Enzymes, Immobilized; D005971:Glutamates; D005973:Glutamine; D018698:Glutamic Acid; D006861:Hydrogen Peroxide; D000594:Amino Acid Oxidoreductases; C023061:L-glutamate oxidase; D005972:Glutaminase