Investigation of experimental factors in in-capillary continuous enzyme assay to obtain plateau response by electrophoretically mediated microanalysis.

In-capillary enzymatic reaction was investigated in electrophoretically mediated microanalysis to achieve plateau response of the reaction product. Hydrolysis of 4-nitrophenyl phosphate (NPP) with alkaline phosphatase (ALP) was used as a model enzymatic reaction. The reaction product of 4-nitrophenol (NP) was photometrically detected as a plateau response based on the continuous enzymatic reaction in the separation capillary and the ongoing electrophoretic resolution of the product from the reaction zone. Two types of injection were compared between an enzyme injection and a substrate injection, whereas the counterpart reagent was contained in the separation buffer. In both approaches, the plateau response of NP developed linearly with increasing sample injection period, as well as the enzyme concentration. At low separation voltages, NP accumulated at the reaction zone, and the plateau height was inversely proportional to the applied voltage. Comparing enzyme and substrate injection methods, a stable plateau response was more easily achieved with the enzyme injection. It is because the concentration of the substrate in the injected plug decreases during the electrophoresis, leading to a reduced reaction rate. In contract, enzyme injection maintains consistent reaction conditions. The Michaelis-Menten constant was determined with the plateau response by both injection modes, and the values were comparable with each other.