The aim of this study is to find optima of operating conditions of phosphorylation enzymatic reaction of glucose within differential pH sensors device. Five variables were studied, i.e., amount of enzyme (7.45-44.7 µl), buffer concentration (20-50mM), pH of buffer (6.8 to 8.1), ATP concentration (0.2-2mM) and Mg +2 concentration (1.2 to 6mM).The kinetic study indicated optima of amount of enzyme of (30µl), buffer concentration of (40mM), pH value of buffer of (7.6), ATP concentration of (1.2mM) and Mg 2+ concentration of (2.2mM) for phosphorylation of 1g/l glucose concentration sample. A calibration curve of glucose quantification was done for low glucose concentration range i.e. from 0 to 1 g/l. This low range makes the assay of this study efficient to be used in many applications. For instance, the assay can be used in glucose quantification during cultivation of variety kinds of cells at low glucose concentration. The assay was developed by using HEPES buffer as new carrying buffer system.
A differential pH measurement device was used to achieve operation conditions of alcohol dehydrogenase reaction. Optimum operating conditions were temperature of 30°C, 10 µl of alcohol dehydrogenase enzyme volume (with a final activity of 563.75 units ml-1) per 50 µl of sample, NAD + concentration of 0.05 mM and 20 mM glycine-pyrophosphate buffer solution of pH 9.1. In this method a range of ethanol concentrations from 0 -99,985 %, which means 0.000001714 -17.14 M, were used. The maximum obtained change in pH, delta pH, was (-33) mpH. A calibration curve of logarithmic values of ethanol concentrations against change in pH for standard ethanol samples was done. Since this calibration curve is a linear with a correlation coefficient (R) of 0.998, this calibration curve can be used in quantification of ethanol concentration. End point of equilibrium concentrations of reactants and products of ethanol oxidation reaction was measured within spectrophotometer. The results indicated 100 seconds of process time is required to reach the end point for all ethanol standard samples. This required time was satisfied with results of measuring change in pH within differential pH analyzer system.
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