Selective and sensitive biosensor for theophylline based on xanthine oxidase electrode

Milk and microbial xanthine oxidases (XOs) were used for the construction o f amperometric enzyme electrodes. Substrate specificity differences of thes e enzymes were studied. Of the two enzymes, only the microbial XO was found to oxidize theophylline, but not theobromine and caffeine. The substrate s pecificity of microbial XO was affected by pH, where the optimum for xanthi ne was 5.5, while for theophylline it was in the range from 6.5 to 8.5. The theophylline biosensor showed a low detection limit of 2 x 10(-7) M and si gnal linearity up to 5 x 10(-5) M. The sensitivity of the microbial XO elec trode to theophylline could be selectively eliminated by immersion in alkal ine phosphate solution, thus allowing for the construction of a blank elect rode for differential measurements. The feasibility of this approach has be en demonstrated by the determination of free (unbound) and total theophylli ne in blood samples. The biosensor exhibited good operational (>6 h) and sh elf (>3 months) stability when trehalose was used as a stabilizer of the bi ocatalytic layer. (C) 2000 Academic Press.