Fabrication of a bilayer potentiometric phosphate biosensor by cross-link immobilization with bovine serum albumin and glutaraldehyde

Chemical cross-linking of purine nucleoside phosphorylase (PNP) and xanthine oxidase (XOD) with glutaraldehyde (GLA) and bovine serum albumin (BSA) has been used to fabricate a stable and reliable bilayer potentiometric phosphate biosensor. The bilayer arrangement consists of an inner BSA-GLA layer and an outer BSA-GLA-PNP-XOD layer. The inclusion of the inner BSA-GLA layer improves the adhesion of the outer BSA-GLA-PNP-XOD layer and ensures stability of the phosphate biosensor. Established optimum conditions for immobilization of the enzymes in the outer layer and for reliable potentiometric measurement were 4.5% v/v GLA, 6.8% w/v BSA, XOD:PNP mole ratio of 1:8, and a film drying time of 30 min. As little as 20 mu M of phosphate can be detected with the BSA-GLA/BSA-GLA-XOD-PNP bilayer biosensor with a linear concentration range between 40 and 120 mu M. The biosensor was very stable for 21 days, achieving a good reproducibility with a rsd of only 5.7% and, even after more than a month, the change in the initial potential value was only 10%. (C) 2011 Elsevier B.V. All rights reserved.