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.

[1]  Chen Chien-Yuan,et al.  Amperometric L-glutamate sensor using a novel L-glutamate oxidase from Streptomyces platensis NTU 3304 , 1991 .

[2]  H. Yoshino,et al.  Purification and Properties of a New Enzyme, L-Glutamate Oxidase, from Streptomyces sp. X-119-6 Grown on Wheat Bran , 1983 .

[3]  J. Kittrell,et al.  Deactivation studies of immobilized glucose oxidase , 1978 .

[4]  H. Müller,et al.  A specific enzyme electrode for l-glutamate-development and application , 1989 .

[5]  Charles L. Cooney,et al.  L-Glutamine Enzyme Electrode for On-Line Mammalian Cell Culture Process Control , 1987 .

[6]  H. Blanch,et al.  Transient responses of hybridoma cells to lactate and ammonia pulse and step changes in continuous culture , 1988 .

[7]  M. Butler.  Growth limitations in high density microcarrier cultures. , 1985, Developments in biological standardization.

[8]  W. Thilly,et al.  High yields from microcarrier cultures by medium perfusion. , 1983, Journal of cell science.

[9]  A. Sinskey,et al.  Mammalian cell culture: engineering principles and scale-up , 1983 .

[10]  T. Chang,et al.  Conversion of α‐ketoglutarate into L‐glutamic acid with urea as ammonium source using multienzyme systems and dextran‐NAD+ immobilized by microencapsulation within artificial cells in a bioreactor , 1988, Biotechnology and bioengineering.

[11]  S. Hartman.  Glutaminase of Escherichia coli. I. Purification and general catalytic properties. , 1968, The Journal of biological chemistry.

[12]  J H Luong,et al.  Development of a biosensor for assaying postmortem nucleotide degradation in fish tissues , 1990, Biotechnology and bioengineering.

[13]  R. Dean,et al.  Effects of exogenous amines on mammalian cells, with particular reference to membrane flow. , 1984, The Biochemical journal.