Ultrafast Formation of Microdroplet Arrays with Chemical Gradients for Label-Free Determination of Enzymatic Reaction Kinetics

Abstract

High throughput assays including enzymatic reactions are usually conducted in multiwell plates and analyzed in plate readers. This approach has limitations in i) upscaling and ii) the choice of reactions, as labeled compounds are required. A technique is introduced to rapidly generate dense microdroplet arrays by stream shearing (MASS). The fluid is delivered via a capillary onto a glass plate that carries a pattern of hydrophilic spots surrounded by a hydrophobic coating. Moving the glass plate shears off droplets from the fluid stream that are retained on the hydrophilic spot. Arrays of up to 24 192 homogenous droplets (coefficient of variation: 2.9 %) with defined volumes from 280 to 980 pL are generated in less than 22 min. Thereby, the droplet content is varied, and fine chemical gradients are obtained across the plate, which are analyzed with both fluorescence microscopy and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). This method is employed for label-free kinetic studies of an enzymatic reaction. For the cleavage of angiotensin II-fluorescein by the angiotensin II-converting enzyme, a maximum reaction velocity (v_max) of 5.7 µm min⁻¹ is determined and the Michaelis-Menten constant (K_M) of 84.8 µm is found. The platform can be further upscaled for biochemical assays as required for drug discovery and protein engineering.