Development of a microplate reader compatible microfluidic device for enzyme assay

Abstract

We report a novel platform technology of enzyme assay using microfluidic channel. Employing the patterns of microfluidic channel on a plastic chip, the dilution of substrate solution and enzyme reactions are sequentially carried out in microfluidic channels, The microfluidic device is fabricated by micromolding processes with polydimethylsiloxane (PDMS) polymer. The device consists of microfluidic channel modules running four assay tests on a single device and each module comprises six reaction chambers linked with microfluidic channels. Overall dimension of the microfluidic device is the same size as a 96-well microtiter plate and 24 reaction chambers in device adapt to the conventional 96-well architecture to be detected by a microplate reader. This provides the method to overcome the limitations of conventional assay platform which requires a special detection instrument, the manual dilution of the substrate solution and individual measurement of enzyme reactions. Enzyme reactions simultaneously occur in six reaction chambers of each microfluidic assay module filled with different concentrations of substrate solution generated by microfluidic channel network. Enzyme activity is measured with continuous methods and is determined by V-max obtained by Michaelis-Menten relationship. In this study. alkaline phosphatase (ALP) and p-nitrophenyl phosphate (p-NPP) have been used as an enzyme-substrate model system and demonstrated for microfluidic enzyme assay experiments.