Authors: Hala Nehmé Reine Nehmé Pierre Lafite Eric Duverger Sylvain Routier Philippe Morin
Publish Date: 2013/09/22
Volume: 405, Issue: 28, Pages: 9159-9167
Abstract
In this study a novel capillary electrophoresis CEbased enzymatic assay was developed to evaluate enzymatic activity in whole cells βGalactosidase expression was used as an example as it is a biomarker for assessing replicative senescence in mammalian cells It catalyzes the hydrolysis of paranitrophenylβdgalactopyranoside PNPG into paranitrophenol PNP The CEbased assay consisted of four main steps 1 hydrodynamic injection of whole intact cells into the capillary 2 incapillary lysis of these cells by using pulses of electric field electroporation 3 incapillary hydrolysis of PNPG by the βgalactosidase—released from the lysed cells—by the electrophoretically mediated microanalysis EMMA approach and 4 online detection and quantification of the PNP formed The developed method was applied to Escherichia coli as well as to human keratinocyte cells at different replicative stages Results obtained by CE were in excellent agreement with those obtained from offline cell lysates which proves the efficiency of the incapillary approach developed This work shows for the first time that cell membranes can be disrupted incapillary by electroporation and that the released enzyme can be subsequently quantified in the same capillary Enzyme quantification in cells after their incapillary lysis has never been conducted by CE The developed CE approach is automated economic ecofriendly and simple to conduct It has attractive applications in bacteria or human cells for early disease diagnostics or insights for development in biology
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