Determination of Kinetic Parameters, Km and kcat, with a Single Experiment on a Chip

We have demonstrated a multistep enzyme reaction on a chip to determine the key kinetic parameters of enzyme reaction. We designed and fabricated a fully integrated microfluidic chip to have sample metering, mixing, and incubation functionalities. The chip generates a gradient of reagent concentrations in 11 parallel processors. We used beta-galactosidase and its substrate, resorufin-beta-D-galactopyranoside, as the model system of the enzyme reaction. With, a single experiment on the chip, we determined the key parameters for the enzyme kinetics, K-m and k(cat), and evaluated the effect of inhibitor concentrations on the reaction rates. This study provides a new tool for evaluating various effectors, such as inhibitors and cofactors, on the initial rate of an enzyme reaction, and it could be applied to a comprehensive bio/chemical reaction study.