Activity and stability of urease enzyme molecules for on-site urea measurement in milk

Abstract: The nitrogenous metabolite urea is an important biomarker that indicates cows’ nutritional intake. Today, determining the urea concentration from milk samples requires analysis techniques in a lab which is time-consuming and expensive. It would be favourable to have an on-site measurement method to achieve a fast detection allowing farmers to quickly adjust the feed of individual cows, which might make it possible to reduce costs, increase milk production, and/or reduce the amount of nitrogen emission to the environment. This thesis is a part of a collaboration project between Linköping University and the Swedish University of Agricultural Sciences named “On-farm measurement of milk urea - development of a sensor”. The thesis investigated the activity and stability of the urease in an electrochemical biosensor, in which the urease is immobilised via encapsulation in a gel of poly(carbamoyl sulfonate) on a screenprinted electrode coated with a metal catalyst (copper), a cation exchanger (Nafion) and a conductive polymer (polyaniline). The linear range of the biosensor was successfully extended up to 2500 μM urea with a diffusion barrier composed of chitosan and polyvinyl butyral, enabling higher urea concentrations measurement than without the barrier (680 μM). Reproducibility, reusability, and storage stability measurements of the urease immobilised electrodes were performed and evaluated. A comparison between free enzyme, immobilised enzyme on the electrode surface and immobilised enzyme on an electrode surface that had been stored for a few days, was conducted to determine urease activity and stability. In addition, five components of milk (casein, lactose, ammonium, iron, and ascorbic acid) were measured separately to evaluate their interferences during milk urea detection. Interference was observed in several cases. A final evaluation of the present electrochemical biosensor was done by analysis of real milk samples giving promising results for future development.