Stabilised enzyme based diagnostic systems
The detection and quantitation of substances using analytical techniques is an important area in many fields. Accuracy,dependability, reproducibility, specificity and stability of the techniques used are all important. The first three parameters are largely operator dependent, however, specificity and stability of the components of the tests also play a part. The specificity of analysis may be determined chemically or biologically by using enzymes, immunological reagents or receptors of some sort. However, biological molecules are often unstable in purified, isolated forms and must be stabilised in some way to retain activity. The work reported here attempts to increase the knowledge of enzyme stabilisation, using the enzyme alcohol oxidase as a test enzyme. This enzyme was used in : (i) A manual assay for ethanol determination. (ii) An automated assay using both soluble and immobilised enzyme in segmented flow and flow injection analysis. (iii) A dry phase stabilised enzyme based test for ethanol in saliva. During the course of the work a method for stabilising the enzyme was discovered. This has been applied to a number of other enzyme systems, successfully stabilising them in most cases. This work forms the basis of a patent application for enzyme stabilisation. A novel detection system allowing semi-quantitative results to be obtained, using stabilised dry phase technology has also been discovered. A second patent application has been filed and the system has been applied to analytes measured by oxidase enzymes. The final area of investigation was to develop an enzymic assay for diacetyl. This substance is a contaminant in beer and as such requires accurate detection at low levels. The purification and characterisation of diacetyl reductase from a new source, (chicken liver) enabled various assay formats to be investigated. These included dye linked assays and enzyme amplified recycling assays to determine diacetyl in aqueous samples.