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 μM of phosphate can be detected with the BSA–GLA/BSA–GLA–XOD–PNP bilayer biosensor with a linear concentration range between 40 and 120 μM. 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%.