Glutamine biosensors for biotechnology applications, with suppression of the endogenous glutamate signal.
Glutamine membranes for amperometric measurements are described. The interference of the endogenous glutamate is greatly diminished by using a supplementary "anti-glutamate" layer consisting of immobilized glutamate oxidase and catalase on top of the glutamine-sensitive layer having co-immobilized glutaminase and glutamate oxidase. The use of polycarbonate membranes with different permeability characteristics for the control of the substrate's access to the enzyme layers is presented, as well as the effect of the density of the enzyme layer on the sensitivity of these membranes. The fabricated membranes have good operational stability (at least 5 days) and very good linearity (up to 10 mM glutamine). Using an appropriate choice of membranes and cross-linking conditions, membranes with good rejection of glutamate have been fabricated (less than 6% RSD for a 5 mM glutamine sample containing 5 mM glutamate as interferent). These membranes are suitable for monitoring of glutamine levels in mammalian cell cultures without the need of a separate measurement for glutamate.
Mădăraş
M B
MB
Department of Chemistry, University of North Carolina at Chapel Hill 27599-3290, USA. mmadaras@ysi.com
Spokane
R B
RB
Johnson
J M
JM
Woodward
J R
JR
D000594:Amino Acid Oxidoreductases / Q000378:metabolism; D015374:Biosensing Techniques; D002374:Catalase / Q000378:metabolism; D004800:Enzymes, Immobilized / Q000378:metabolism; D018698:Glutamic Acid / Q000032:analysis*; D005973:Glutamine / Q000032:analysis*
D004800:Enzymes, Immobilized; D005973:Glutamine; D018698:Glutamic Acid; D002374:Catalase; D000594:Amino Acid Oxidoreductases; C023061:L-glutamate oxidase