2026 Volume 47 Issue 1 Pages 63-70
In-capillary continuous enzyme assay in electrophoretically mediated microanalysis was simulated using MATLAB software, employing the hydrolysis of p-nitrophenylphosphate (NPP) with alkaline phosphatase (ALP) as a model system. In the simulation, a short plug of NPP substrate solution was injected into a separation capillary, while the separation buffer contained the enzyme ALP. The migration of NPP in the capillary was modeled based on its apparent electrophoretic mobility, and enzymatic hydrolysis of NPP occurred continuously within the NPP zone at a defined reaction rate. A product of p-nitrophenol (NP) was continuously generated at the substrate zone, and it was immediately resolved from the substrate zone with its apparent electrophoretic mobility. Electrophoretic migration and enzymatic reaction were iterated every second, resulting in a simulated plateau response of NP in the continuous enzyme assay. Electropherograms exhibiting the plateau response were reproduced by adjusting several parameters, including instrumental conditions (capillary lengths, injected plug length, applied voltage), reaction conditions (reagent concentrations, reaction rate), and electrophoretic mobility of both the substrate and the product. The height of the plateau response reflected the Michaelis-Menten constant and the reaction kinetics, and the simulation results showed good agreement with previously reported experimental data. At low substrate concentrations, the plateau response transitioned to a slope profile, indicating significant substrate depletion. Additional parameter sets were explored to achieve the plateau response. This study demonstrates that MATLAB-based simulation is a helpful tool for estimating the conditions to achieve a plateau response in electrophoretically mediated microanalysis.
