Miniaturized amperometric biosensor based on xanthine oxidase for monitoring hypoxanthine in cell culture media

Fabrication and characterization of miniaturized amperometric hypoxanthine biosensors are described and demonstrated for monitoring hypoxanthine in my ocardial cell culture media. The sensors are based on xanthine oxidase (XO) immobilized on carbon fiber microelectrodes (CFMEs) using a composite film of Nafion and electropolymerized phenol (PPh), Nafion was used for XO immo bilization because of its film hydrophobicity, enzyme-favored environment., and electrostatic interaction with XO, which was dispersed in Nafion film by immersing the Nafion-coated CFMEs in XO solution for 5 h, PPh film was f ormed as an overlay on Nafion and XO-modified CFMEs via electropolymerizati on. Hypoxanthine was measured with the sensor by the oxidation of enzymatic reaction products, hydrogen peroxide (H2O2), and uric acid (UA) at +0.60 V (vs Ag/AgCl). The use of Nafion and PPh as a matrix for XO immobilization yields enhanced specificity, sensitivity, and linearity toward hypoxanthine . A dynamic linear range of 5.0 muM to 1.8 mM was achieved with a calculate d detection limit of 1.5 muM (S/N = 3) and a sensitivity of 3.144 nA/mM. In addition, the measurement was virtually interference-free from easily oxid izable species such as UA, ascorbic acid, physiological levels of neurotran smitters, and their principal metabolites. The biosensor was used to monito r hypoxanthine accumulation in myocardial cell culture media, in which the level of extracellular hypoxanthine was found to increase with ischemic tol erance, (C) 2001 Academic Press.