Study of an enzyme coupled system for the development of fibre optical bilirubin sensors

We have established an analytical procedure for bilirubin (BR) determination by following the variation of its yellow absorption band in the presence of the biocatalytic system haemoglobin/glucose oxidase/glucose. The influence of the pH of the medium, the haemoglobin/hydrogen peroxide ratio, and the concentration of the various components in the mixture on the system activity was evaluated. The solubility of the unconjugated BR was analysed. An anionic detergent in Tris buffer, in which it presents a high critical micellar concentration, was selected for the BR/bovine serum albumin cleavage. The Michaelis-Menten constant of the system (after the optimisation of the various parameters) was determined and compared with Km values reported in the literature for similar catalytic systems. Preliminary results obtained with the haemoglobin and glucose oxidase immobilised either in poly(vinyl alcohol) or by crosslinking with glutaraldehyde, show that this system is promising as a future biosensor.