Single-Shot Characterization of Enzymatic Reaction Constants Km and kcat by an Acoustic-Driven, Bubble-Based Fast Micromixer

In this work we present an acoustofluidic approach for rapid, single-shot characterization of enzymatic reaction constants K-m and k(cat). The acoustofluidic design involves a bubble anchored in a horseshoe structure which can be stimulated by a piezoelectric transducer to generate vortices in the fluid. The enzyme and substrate can thus be mixed rapidly, within 100 ms, by the vortices to yield the product. Enzymatic reaction constants K-m and k(cat) can then be obtained from the reaction rate curves for different concentrations of substrate while holding the enzyme concentration constant. We studied the enzymatic reaction for beta-galactosidase and its substrate (resorufin-beta-D-galactopyranoside) and found K-m and k(cat) to be 333 +/- 130 mu M and 64 +/- 8 s(-1), respectively, which are in agreement with published data. Our approach is valuable for studying the kinetics of high-speed enzymatic reactions and other chemical reactions.