Probing enzymatic activity inside single cells.
Journal article, 2013

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.

Author

Jessica Olofsson

Chalmers, Chemical and Biological Engineering

Stanford University

Xu Shijun

Chalmers, Chemical and Biological Engineering, Physical Chemistry

Gavin Jeffries

Chalmers, Chemical and Biological Engineering, Physical Chemistry

Aldo Jesorka

Chalmers, Chemical and Biological Engineering, Physical Chemistry

Helen Bridle

Physical Chemistry

Heriot-Watt University

Ida Isaksson

Chalmers, Chemical and Biological Engineering, Physical Chemistry

S. G. Weber

University of Pittsburgh

Owe Orwar

Chalmers, Chemical and Biological Engineering, Physical Chemistry

Analytical Chemistry

0003-2700 (ISSN) 1520-6882 (eISSN)

Vol. 85 21 10126-33

Areas of Advance

Nanoscience and Nanotechnology (2010-2017)

Subject Categories

Physical Chemistry

DOI

10.1021/ac4013122

PubMed

24003961

More information

Latest update

1/17/2019