| Alkaline phosphatase is a phospho-monoesterase enzyme, which can catalyze thehydrolysis of monoesters of phosphoric acid (at alkaline pH), and yield phosphate and thecorresponding alcohol. The abundance in nature and importance of this enzyme in biologicalsystems has made ALP activity assessments one of the most commonly performed enzymatictests. Sequential injection analysis (SIA) systems have been extensively exploited in the lastdecades for the implementation of enzyme based assays aiming the evaluation of enzymeactivity or the determination of specific analytes. Based on aforementioned, this thesisexploited two experiments as the following:Part I：Using4-methylumbelliferone phosphate (4-MUP) as the substrate of ALP，aSIA-fluorometry system for the determination of ALP activity was developed. Theexperimental variables, i.e. temperature, pH, the concentration and volume of4-MUP orsamples, the reaction time and the flow rate, were investigated. By loading20μL samples and40μL4-MUP, the calibration curve showed an excellent linearity in the activity range of50~1000mU/L of ALP, the linear regression equation is ΔF=143.4CALP+6.859(r2=0.9988), thedetection limit (3σ) of14mU/L was achieved, along with a sampling frequency of15h-1anda precision of2.3%(200mU/L, n=11).Part II：Endogenous milk ALP manifests a slightly higher heat resistance than thepathogenic microflora upon which pasteurization time and temperature requirements arebased. Hence, ALP activity is recognized and accepted as the method of choice for the rapidvalidation of milk product pasteurization. In this work, the established SIA-fluorometrysystem was applied in the determination of the activity of ALP from milk products. Theresults showed that the present system was an effective platform for evaluation ofpasteurization. |
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