Fabrication of phosphate biosensor by immobilisation of purine nucleoside phosphorylase and xanthine oxidase

Phosphate is one of the major contaminants of waterways such as lakes, rivers and estuaries. Its analysis is of great importance for the prevention of eutrophication of waterways, as well as the prevention of hypothyroidism and bone diseases in humans. Classical methods of analysis, such as spectrophotometry, chromatography and titrimetric methods, have been the usual assay methods, which are time-consuming because of extensive sample preparation. Fabrication of a phosphate biosensor by physical immobilisation of purine nucleoside phosphorylase (PNP) and xanthine oxidase (XOD) in this study is intended to provide a more rapid method of analysis, while also improving the selectivity, accuracy and precision of phosphate measurement. During the investigation of the co-immobilisation of PNP and XOD into polypyrrole (PPy) film, it was found that the XOD immobilised in this polymer is also useful for the determination of hypoxanthine in fish. Subsequent development of a strategy for co-immobilisation of XOD and PNP enabled the determination of phosphate. Some of the important considerations during the development of the phosphate biosensor included establishment of conditions for immobilisation of the enzymes into polypyrrole film, bovin serum albumin (BSA)/glutaraldehyde (GLA) gel and a hybrid bilayer of PPy and BSA-GLA. In order to achieve good resolution, reproducibility and highly sensitive phosphate and hypoxanthine sensors, parameters such as current density, pH, pyrrole concentration, polymerisation and drying time were optimised. Chapter 1 is a literature review, which covers the importance of phosphate, effect of phosphate on the environment, analytical methods for phosphate determination, and use of a biosensor for determination of phosphate. The origins of phosphate and its role in causing disease in the body are discussed. Previous and current chemical methods of phosphate determinations of phosphate are reviewed. Also, the use of a bienzyme system for development of a biosensor for the determination of phosphate is discussed. In Chapter 2 a biosensor is developed for the determination of hypoxanthine by entrapment of XOD and a mediator, ferrocene or potassium ferrocyanide, (~Fe(CN) 6, into a PPy film during galvanostatic film formation. The optimum conditions for the formation of PPy-XOD-Fe(CN)6 4 - film include 0.4 M pyrrole, 6.2 U/ml XOD, 40 mM ~Fe(CN) 6, polymerisation period of 200 seconds, and an applied current density of 0.5 mA/cm2 • Two modes of detection, the potentiometric and amperometric modes, were investigated. The optimum potential for the amperometric biosensing of phosphate was 800 m V vs. Ag/ AgCl (3M KCl) in 0.05 M phosphate buffer. A comparison of the sensitivities for the amperometric and potentiometric modes revealed that potentiometric detection was more sensitive and gave a wider linear concentration range. The PPy-XOD biosensor was successfully used for the determination of fish freshness. In Chapter 3 a biosensor is developed by co-entrapment of XOD, PNP and ~Fe(CN)6 into a PPy film via galvanostatic polymerisation of pyrrole. The optimum conditions for formation of the PPy-PNP-XOD-Fe(CN)6 4- - film are 0.3 M pyrrole. 6.2 U/ml XOD, 49 U/ml PNP, 40 mM ~Fe(CN) 6, polymerisation period of200 seconds and an applied current density of 0.5 mA/cm2 . The optimum potential for the amperometric biosensing of phosphate was 800 mV vs Ag/AgCl (3M KCl) in 0.05 M barbitone buffer. The achievable linear concentration range was between 0.1 and 1 mM, while the minimum detectable amount was 10 J..LM. In Chapter 4 a biosensor is developed, as in Chapter 3, based on potentiometric detection mode. PNP and XOD were immobilised as described in Chapter 3, into polypyrrole films. Potentiometric measurement of phosphate was achieved in 0.05 M barbitone buffer (pH 7.8) and in the presence of 10 mM inosine. A minimum detectable amount of 1 J..tM phosphate and a linear concentration range of 5-20 J..lM were achieved. The objective of the work described in Chapter 5 was to develop an alternative method of enzyme immobilisation in order to improve the sensitivity of the phosphate biosensor. PNP and XOD were immobilised by chemical cross-linking with GLA and BSA. Sensitive potentiometric measurement of phosphate was achieved with 4.5% v/v GLA, 5.8% w/v BSA, XOD:PNP ratio of 1:8 and a film drying time of 30 minutes. The minimum detectable amount was 20 J..tM and the linear range was between 40 and lOOJ..tM. In Chapter 6 a hybrid bilayer biosensor was fabricated with a polypyrrole inner layer and an outer layer where PNP and XOD were chemically cross-linked with BSA and GLA. The best response was obtained when the inner layer was polymerised with 0.01 M KN03 and 0.1 M pyrrole; and 3 J..lL of a mixture ofXOD, PNP, GLA and BSA was spread on top of the PPy electrode to give optimum film thickness. The optimum composition of the second layer was: 4.45% v/v GLA, 6.8 w/v BSA and PNP:XOD ratio of 8:1. The optimum drying time for the outer layer was 20 minutes. A detection limit of 20 J..lM and a linear concentration range of 20-200 J..tM was achieved. The biosensor was used for the determination of phosphate in river and lake water samples.