High‐throughput enzyme kinetics using microarrays

We report a microanalytical method to study enzyme kinetics. The technique involves immobilizing horseradish peroxidase on a poly‐L‐lysine (PLL)‐coated glass slide in a microarray format, followed by applying substrate solution onto the enzyme microarray. Enzyme molecules are immobilized on the PLL‐coated glass slide through electrostatic interactions, and no further modification of the enzyme or glass slide is needed. In situ detection of the products generated on the enzyme spots is made possible by monitoring the light intensity of each spot using a scientific‐grade charged‐coupled device (CCD). Reactions of substrate solutions of various types and concentrations can be carried out sequentially on one enzyme microarray. To account for the loss of enzyme from washing in between runs, a standard substrate solution is used for calibration. Substantially reduced amounts of substrate solution are consumed for each reaction on each enzyme spot. The Michaelis constant K m obtained by using this method is comparable to the result for homogeneous solutions. Absorbance detection allows universal monitoring, and no chemical modification of the substrate is needed. High‐throughput studies of native enzyme kinetics for multiple enzymes are therefore possible in a simple, rapid, and low‐cost manner.