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

Abstract

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 β-galactosidase and its substrate (resorufin-β-D-galactopyranoside) and found K_m and k_cat to be 333 ± 130 μM 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.