Probing enzymatic activity inside single cells.
Artikel i vetenskaplig tidskrift, 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.

Författare

Jessica Olofsson

Stanford University School of Medicine

Chalmers University of Technology

Xu Shijun

Chalmers, Kemi- och bioteknik, Fysikalisk kemi

Gavin Jeffries

Chalmers, Kemi- och bioteknik, Fysikalisk kemi

Aldo Jesorka

Chalmers, Kemi- och bioteknik, Fysikalisk kemi

Helen Bridle

Heriot-Watt University, Edinburgh

Chalmers University of Technology

Ida Isaksson

Chalmers University of Technology

S. G. Weber

University of Pittsburgh

Owe Orwar

Chalmers, Kemi- och bioteknik, Fysikalisk kemi

Analytical Chemistry

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

Vol. 85 10126-33

Styrkeområden

Nanovetenskap och nanoteknik

Ämneskategorier

Fysikalisk kemi

DOI

10.1021/ac4013122

PubMed

24003961