title: High-throughput, quantitative enzyme kinetic analysis in microdroplets using stroboscopic epifluorescence imaging.
abstract: Droplet-based microfluidic systems offer a range of advantageous features for the investigation of enzyme kinetics, including high time resolution and the ability to probe extremely large numbers of discrete reactions while consuming low sample volumes. Kinetic measurements within droplet-based microfluidic systems are conventionally performed using single point detection schemes. Unfortunately, such an approach prohibits the measurement of an individual droplet over an extended period of time. Accordingly, we present a novel approach for the extensive characterization of enzyme-inhibitor reaction kinetics within a single experiment by tracking individual and rapidly moving droplets as they pass through an extended microfluidic channel. A series of heterogeneous and pL-volume droplets, containing varying concentrations of the fluorogenic substrate resorufin β-d-galactopyranoside and a constant amount of the enzyme β-galactosidase, is produced at frequencies in excess of 150 Hz. By stroboscopic manipulation of the excitation laser light and adoption of a dual view detection system, "blur-free" images containing up to 150 clearly distinguishable droplets per frame are extracted, which allow extraction of kinetic data from all formed droplets. The efficiency of this approach is demonstrated via a Michaelis-Menten analysis which yields a Michaelis constant, Km, of 353 μM. Additionally, the dissociation constant for the competitive inhibitor isopropyl β-d-1-thiogalactopyranoside is extracted using the same method.
authors: Institute for Chemical and Bioengineering, Department of Chemistry and Applied Biosciences, ETH Zürich, Vladimir Prelog Weg 1, 8093 Zürich, Switzerland.
authors: David
authors: D
authors: Hess
authors: Institute for Chemical and Bioengineering, Department of Chemistry and Applied Biosciences, ETH Zürich, Vladimir Prelog Weg 1, 8093 Zürich, Switzerland.
authors: Anandkumar
authors: A
authors: Rane
authors: Institute for Chemical and Bioengineering, Department of Chemistry and Applied Biosciences, ETH Zürich, Vladimir Prelog Weg 1, 8093 Zürich, Switzerland.
authors: Andrew J
authors: AJ
authors: deMello
authors: Institute for Chemical and Bioengineering, Department of Chemistry and Applied Biosciences, ETH Zürich, Vladimir Prelog Weg 1, 8093 Zürich, Switzerland.
authors: Stavros
authors: S
authors: Stavrakis
mesh_terms: D004789:Enzyme Activation; D057075:Enzyme Assays / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology; D057166:High-Throughput Screening Assays; D007700:Kinetics; D007834:Lasers; D046210:Microfluidic Analytical Techniques; D010316:Particle Size; D013499:Surface Properties; D001616:beta-Galactosidase / Q000032:analysis* / Q000378:metabolism*
chemical_list: D001616:beta-Galactosidase