Comparison of enzyme immobilisation methods for potentiometric phosphate biosensors

The development of two phosphate biosensors is described and compared for potentiometric detection of phosphate. Purine nucleoside phosphorylase (PNP) and xanthine oxidase (XOD) were co-immobilised by chemical cross-linking with glutaraldehyde (GLA) and bovine serum albumin (BSA), and via entrapment into polypyrrole (PPy) films by galvanostatic polymerisation. The BSA-GLA film was made with 4.5% v/v GLA and 6.8% w/v BSA with a drying time of 30min, while polypyrrole entrapment was achieved with 0.5M pyrrole by using a polymerisation time of 200s. A mole ratio of 1:8 (6.2U/mL XOD: 49.6U/mL PNP) was used for both methods of enzyme immobilisation. Sensitive potentiometric measurements obtained for phosphate with the BSA-GLA-PNP-XOD biosensor were compared with those of PPy-PNP-XOD-Fe(CN)64− biosensor. A minimum detectable concentration of 0.1mg/L phosphate and a linear concentration range of 0.5–2.5mg/L were achieved with the PPy-PNP-XOD-Fe(CN)64− biosensor. In comparison, a minimum detectable concentration of 2mg/L and a linear concentration range of 4–12mg/L were achieved with the BSA-GLA immobilisation. The presence of uric and ascorbic acids had the least effect on the performance of the PPy-PNP-XOD-Fe(CN)64− biosensor, but will not have any effect on phosphate measurement with both biosensors at levels normally present in water.