Amperometric biosensors for detection of choline and lactate by Choline Oxidase and Lactate Oxidase immobilisation in microporous membranes

This project report deals with the construction of amperometric biosensors for the detection of choline and lactate, using Choline Oxidase and Lactate Oxidase as the selective element. The enzymes were immobilised in a variety of microporous membrane supports using different immobilisation protocols. Microporous membranes for immobilisation of biologically active materials are available commercially from Millipore (USA) as "Immobilon" and Pall (UK) as Pall "Biodyne". Also "track-etch" membranes (Cyclopore SA, Belgium) were used in the comparison as a promising new support. The electrochemical sensor response of the membranes was compared with a spectrophotometric determination of enzyme activity for the various immobilisation methods used. The best membrane for immobilisation appeared to be the track-etch membrane, which gave a fast response and a good reproducibility. The problem with these membranes, however, in the case of ChOx immobilisation was the saturation of the choline response in the measurement range. Generally, the Immobilon and Pall membranes have a larger binding capacity, but give lower reproducibility as the track-etch membranes. A more designed approach to effective immobilisation is in principle possible when the crystal structure of an enzyme is available. The exact structure of lactate oxidase, however, is still unknown. In this study the immobilisation of enzymes has been studied merely in an empirical fashion. The large differences observed with various methods and membranes were quite striking and highlight the importance of choosing the right materials and methods for immobilisation of enzymes.