title: New generation of amino coumarin methyl sulfonate-based fluorogenic substrates for amidase assays in droplet-based microfluidic applications.
abstract: Droplet-based microfluidics is a powerful tool for biology and chemistry as it allows the production and the manipulation of picoliter-size droplets acting as individual reactors. In this format, high-sensitivity assays are typically based on fluorescence, so fluorophore exchange between droplets must be avoided. Fluorogenic substrates based on the coumarin leaving group are widely used to measure a variety of enzymatic activities, but their application in droplet-based microfluidic systems is severely impaired by the fast transport of the fluorescent product between compartments. Here we report the synthesis of new amidase fluorogenic substrates based on 7-aminocoumarin-4-methanesulfonic acid (ACMS), a highly water-soluble dye, and their suitability for droplet-based microfluidics applications. Both substrate and product had the required spectral characteristics and remained confined in droplets from hours to days. As a model experiment, a phenylacetylated ACMS was synthesized and used as a fluorogenic substrate of Escherichia coli penicillin G acylase. Kinetic parameters (k(cat) and K(M)) measured in bulk and in droplets on-chip were very similar, demonstrating the suitability of this synthesis strategy to produce a variety of ACMS-based substrates for assaying amidase activities both in microtiter plate and droplet-based microfluidic formats.
authors.forename: Gabrielle
authors.initials: G
authors.lastname: Woronoff
authors.forename: Abdeslam
authors.initials: A
authors.lastname: El Harrak
authors.forename: Estelle
authors.initials: E
authors.lastname: Mayot
authors.forename: Olivier
authors.initials: O
authors.lastname: Schicke
authors.forename: Oliver J
authors.initials: OJ
authors.lastname: Miller
authors.forename: Patrice
authors.initials: P
authors.lastname: Soumillion
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Griffiths
authors.forename: Michael
authors.initials: M
authors.lastname: Ryckelynck
mesh_terms: D003374:Coumarins / Q000138:chemical synthesis / Q000737:chemistry*; D057075:Enzyme Assays / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D007700:Kinetics; D008698:Mesylates / Q000138:chemical synthesis / Q000737:chemistry*; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D008958:Models, Molecular; D015394:Molecular Structure; D010399:Penicillin Amidase / Q000032:analysis* / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D003374:Coumarins; D005456:Fluorescent Dyes; D008698:Mesylates; C059117:7-aminocoumarin-4-methanesulfonic acid; D010399:Penicillin Amidase