Probing enzymatic activity inside single cells

Jessica Olofsson, Shijun Xu, Gavin D M Jeffries, Aldo Jesorka, Helen Bridle, Ida Isaksson, Stephen G. Weber, Owe Orwar

Research output: Contribution to journalArticle

5 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)10126-10133
Number of pages8
JournalAnalytical Chemistry
Volume85
Issue number21
DOIs
Publication statusPublished - 4 Sep 2013

ASJC Scopus subject areas

  • Analytical Chemistry

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