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Electrophoretically mediated microanalysis for in-capillaryelectrical cell lysis and fast enzyme quantification by capillary electrophoresis

Analytical and Bioanalytical Chemistry volume 405, pages 9159–9167 (2013)Cite this article

Abstract

In this study, a novel capillary electrophoresis (CE)-based enzymatic assay was developed to evaluate enzymatic activity in whole cells. β-Galactosidase expression was used as an example, as it is a biomarker for assessing replicative senescence in mammalian cells. It catalyzes the hydrolysis of para-nitrophenyl-β-d-galactopyranoside (PNPG) into para-nitrophenol (PNP). The CE-based assay consisted of four main steps: (1) hydrodynamic injection of whole intact cells into the capillary, (2) in-capillary lysis of these cells by using pulses of electric field (electroporation), (3) in-capillary hydrolysis of PNPG by the β-galactosidase—released from the lysed cells—by the electrophoretically mediated microanalysis (EMMA) approach, and (4) on-line 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 off-line cell lysates which proves the efficiency of the in-capillary approach developed. This work shows for the first time that cell membranes can be disrupted in-capillary by electroporation and that the released enzyme can be subsequently quantified in the same capillary. Enzyme quantification in cells after their in-capillary lysis has never been conducted by CE. The developed CE approach is automated, economic, eco-friendly, and simple to conduct. It has attractive applications in bacteria or human cells for early disease diagnostics or insights for development in biology.

Electropherograms for in-capillary reaction catalyzed by β-galactosidase obtained from off-capillary and in-capillary lysis of E. coli cells.

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Abbreviations

BCA:

bicinchoninic acid

BGE:

background electrolyte

CE:

capillary electrophoresis

CPA:

corrected peak area

EMMA:

electrophoretically mediated microanalysis

β-Gal:

β-galactosidase

PNP:

4-nitrophenol

PNPG:

4-nitrophenyl-d-galactopyranoside

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  1. Institut de Chimie Organique et Analytique (ICOA), Université d’Orléans - CNRS FR 2708 - UMR 7311, BP 6759, rue de Chartres, 45067, Orléans cedex 2, France

    Hala Nehmé, Reine Nehmé, Pierre Lafite, Eric Duverger, Sylvain Routier & Philippe Morin

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  1. Hala Nehmé
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  2. Reine Nehmé
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  3. Pierre Lafite
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Correspondence to Reine Nehmé.

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Nehmé, H., Nehmé, R., Lafite, P. et al. Electrophoretically mediated microanalysis for in-capillaryelectrical cell lysis and fast enzyme quantification by capillary electrophoresis. Anal Bioanal Chem 405, 9159–9167 (2013). https://doi.org/10.1007/s00216-013-7332-0

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  • DOI: https://doi.org/10.1007/s00216-013-7332-0

Keywords

  • Capillary electrophoresis
  • Electrophoretically mediated microanalysis
  • Enzyme kinetics
  • β-Galactosidase
  • In-capillary cell lysis
  • Senescence