title: Probing enzymatic activity inside single cells.
abstract: We report a novel approach for determining the enzymatic activity within a single suspended cell. Using a steady-state microfluidic delivery device and timed exposure to the pore-forming agent digitonin, we controlled the plasma membrane permeation of individual NG108-15 cells. Mildly permeabilized cells (~100 pores) were exposed to a series of concentrations of fluorescein diphosphate (FDP), a fluorogenic alkaline phosphatase substrate, with and without levamisole, an alkaline phosphatase inhibitor. We generated quantitative estimates for intracellular enzyme activity and were able to construct both dose-response and dose-inhibition curves at the single-cell level, resulting in an apparent Michaelis contant Km of 15.3 μM ± 1.02 (mean ± standard error of the mean (SEM), n = 16) and an inhibition constant Ki of 0.59 mM ± 0.07 (mean ± SEM, n = 14). Enzymatic activity could be monitored just 40 s after permeabilization, and five point dose-inhibition curves could be obtained within 150 s. This rapid approach offers a new methodology for characterizing enzyme activity within single cells.
authors.affiliation: Department of Chemical and Biological Engineering, Chalmers University of Technology , Kemivägen 10, SE-412 96 Gothenburg, Sweden.
authors.forename: Jessica
authors.initials: J
authors.lastname: Olofsson
authors.forename: Shijun
authors.initials: S
authors.lastname: Xu
authors.forename: Gavin D M
authors.initials: GD
authors.lastname: Jeffries
authors.forename: Aldo
authors.initials: A
authors.lastname: Jesorka
authors.forename: Helen
authors.initials: H
authors.lastname: Bridle
authors.forename: Ida
authors.initials: I
authors.lastname: Isaksson
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Weber
authors.forename: Owe
authors.initials: O
authors.lastname: Orwar
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D002463:Cell Membrane Permeability; D004305:Dose-Response Relationship, Drug; D005453:Fluorescence; D007978:Levamisole / Q000494:pharmacology; D051379:Mice; D009447:Neuroblastoma / Q000201:enzymology* / Q000473:pathology; D051381:Rats; D059010:Single-Cell Analysis
chemical_list: D007978:Levamisole