Kb. Male et al., ONLINE MONITORING OF GLUCOSE IN MAMMALIAN-CELL CULTURE USING A FLOW-INJECTION ANALYSIS (FIA) MEDIATED BIOSENSOR, Biotechnology and bioengineering, 55(3), 1997, pp. 497-504
A flow injection analysis (FIA) biosensor system has been developed fo
r on-line determination of glucose during mammalian cell cultivation.
The culture sample was peristaltically withdrawn from the bioreactor a
nd after cell separation by a steam sterilizable ceramic microfilter,
the filtrate was continuously fed to the FIA mediated-biosensor system
at 4 mLh(-1), whereas the cell-containing retentate was recirculated
to the bioreactor. In the amperometric biosensor system, glucose oxida
se was covalently immobilized onto a preactivated nylon membrane and a
ttached to the sensing area of a platinum working electrode. The enzym
e reaction was coupled with the mediator 1,1'-dimethylferricinium (DMF
e+)-cyclodextrein inclusion complex to recycle the reduced glucose oxi
dase to its original active state. 1,1'- dimethylferrocene (DMFe) was
then reoxidized to DMFe+ at the surface of the platinum electrode pois
ed at + 0.15 V vs silver/silver chloride. The FIA mediated-biosensor w
as linear up to 6 mM glucose, with a detection limit of 0.1 mM, and po
ssessed excellent reproducibility (+/-0.4%, 95% confidence interval) o
ver 123 repeated analyses during a 62 h continuous operation. The immo
bilized glucose oxidase was stable for up to 7 days when applied to gl
ucose measurement during 5-10 day fed-batch cultivation of 293S mammal
ian cells. The results obtained from the mediated-biosensor system com
pared well with the hexokinase and HPLC data. (C) 1997 John Wiley & So
ns.