Date of Award

2004

Document Type

Master Thesis

Degree Name

Masters of Science (Research)

Department

Chemical & Life Sciences

First Advisor

Dr. Marie O'Dowd

Abstract

The thesis focuses on evaluating calcium alginate as an immobilisation matrix for the oxidase enzyme glucose oxidase for the amperometric determination of glucose. The basic principles of enzymology and enzyme immobilisation is discussed followed by a review of a variety of first, second and third generation type enzyme electrodes. Amperometry as governed by the Cottrell equation and mass transfer models relating to amperometric electrodes are also considered.

Bovine serum albumin (BSA / GOx) enzyme electrode system was employed as a model to evaluate a calcium alginate immobilisation technique for glucose oxidase. The principal methods employed for enzyme immobilisation were:

• Bovine serum albumin (BSA) by co-covalent linking of enzyme to protein using the bi-functional reagent glutaraldehyde (chemical cross-linking).

• Sodium alginate by formation of an insoluble gel matrix with the enzyme physically entrapped within. The insoluble alginate gel is formed by substitution of the sodium ions that occur naturally within the gel with calcium ions.

• Calcium alginate by chemical cross-linking of enzyme using the bi-functional reagent glutaraldehyde and polyethylenimine (PEI).

Glucose oxidase entrapped in calcium alginate for glucose analysis in a TRIS-HCl buffer remained stable throughout the testing period. This enzyme electrode gave a response time between 27 and 49 sec, a linear range of up to 10 mM and a sensitivity of 0.13 μA/mM. Kinetic studies revealed that the enzyme electrode was rate limited by enzyme kinetics rather than mass transport, exhibiting an apparent Michaelis constant of Kmapp = 17.6 mM with a limiting current density (Imax) of 1.8 μA. The reproducibility of this enzyme electrode was comparable to that of the model BSA / GOx enzyme electrode system with % RSD ranging from as low as 7.2 % for linearity, 3.3 % for sensitivity and 16.6 % for response time.

A glucose sensitive enzyme electrode was also prepared by chemical cross-linking of glucose oxidase using the bi-functional reagent glutaraldehyde and polyethylenimine (PEI). Unlike the previous system, this electrode could be utilised in a phosphate buffer environment. Although response times were higher than both the model BSA / GOx and Alginate / GOx systems (ca. 1.5 min) the immobilisation procedure was rapid and the resulting electrodes displayed a linear range of up to 23 mM and high sensitivity of 0.17 μA/mM. Average Imax (maximum current) and Kmapp(apparent Michaelis constant) for the optimised PEI / GOx enzyme electrode system were 1.2 μA and 11.6 mM respectively.

Access Level

info:eu-repo/semantics/openAccess

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