title: Development of quantitative cell-based enzyme assays in microdroplets.
abstract: We describe the development of an enzyme assay inside picoliter microdroplets. The enzyme alkaline phosphatase is expressed in Escherichia coli cells and presented in the periplasm. Droplets act as discrete reactors which retain and localize any reaction product. The catalytic turnover of the substrate is measured in individual droplets by monitoring the fluorescence at several time points within the device and exhibits kinetic behavior similar to that observed in bulk solution. Studies on wild type and a mutant enzyme successfully demonstrated the feasibility of using microfluidic droplets to provide time-resolved kinetic measurements.
authors.affiliation: Department of Biochemistry, University of Cambridge, UK.
authors.forename: Ansgar
authors.initials: A
authors.lastname: Huebner
authors.forename: Luis F
authors.initials: LF
authors.lastname: Olguin
authors.forename: Daniel
authors.initials: D
authors.lastname: Bratton
authors.forename: Graeme
authors.initials: G
authors.lastname: Whyte
authors.forename: Wilhelm T S
authors.initials: WT
authors.lastname: Huck
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: de Mello
authors.forename: Joshua B
authors.initials: JB
authors.lastname: Edel
authors.forename: Chris
authors.initials: C
authors.lastname: Abell
authors.forename: Florian
authors.initials: F
authors.lastname: Hollfelder
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002384:Catalysis; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D044085:Microfluidics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: D017931:DNA Primers; D011994:Recombinant Proteins; D000469:Alkaline Phosphatase