title: A fully unsupervised compartment-on-demand platform for precise nanoliter assays of time-dependent steady-state enzyme kinetics and inhibition.
abstract: The ability to miniaturize biochemical assays in water-in-oil emulsion droplets allows a massive scale-down of reaction volumes, so that high-throughput experimentation can be performed more economically and more efficiently. Generating such droplets in compartment-on-demand (COD) platforms is the basis for rapid, automated screening of chemical and biological libraries with minimal volume consumption. Herein, we describe the implementation of such a COD platform to perform high precision nanoliter assays. The coupling of a COD platform to a droplet absorbance detection set-up results in a fully automated analytical system. Michaelis-Menten parameters of 4-nitrophenyl glucopyranoside hydrolysis by sweet almond β-glucosidase can be generated based on 24 time-courses taken at different substrate concentrations with a total volume consumption of only 1.4 μL. Importantly, kinetic parameters can be derived in a fully unsupervised manner within 20 min: droplet production (5 min), initial reading of the droplet sequence (5 min), and droplet fusion to initiate the reaction and read-out over time (10 min). Similarly, the inhibition of the enzymatic reaction by conduritol B epoxide and 1-deoxynojirimycin was measured, and Ki values were determined. In both cases, the kinetic parameters obtained in droplets were identical within error to values obtained in titer plates, despite a >10(4)-fold volume reduction, from micro- to nanoliters.
authors.affiliation: Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Fabrice
authors.initials: F
authors.lastname: Gielen
authors.forename: Liisa
authors.initials: L
authors.lastname: van Vliet
authors.forename: Bartosz T
authors.initials: BT
authors.lastname: Koprowski
authors.forename: Sean R A
authors.initials: SR
authors.lastname: Devenish
authors.forename: Martin
authors.initials: M
authors.lastname: Fischlechner
authors.forename: Joshua B
authors.initials: JB
authors.lastname: Edel
authors.forename: Xize
authors.initials: X
authors.lastname: Niu
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: deMello
authors.forename: Florian
authors.initials: F
authors.lastname: Hollfelder
mesh_terms: D007700:Kinetics; D036103:Nanotechnology / Q000295:instrumentation; D010316:Particle Size; D027861:Prunus / Q000201:enzymology; D013997:Time Factors; D001617:beta-Glucosidase / Q000037:antagonists & inhibitors* / Q000378:metabolism*
chemical_list: D001617:beta-Glucosidase