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.
Similar content being viewed by others
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
References
Cohen D, Dickerson JA, Whitmore CD, Turner EH, Palcic MM, Hindsgaul O, Dovichi NJ (2008) Annu Rev Anal Chem 1:165–190
Gary RK, Kindell SM (2005) Anal Biochem 343:329–334
Herwig E, Marchetti-Deschmann M, Wenz C, Rüfer A, Allmaier G (2011) Biotechnol J 6:420–427
Lidstrom ME, Meldrum DR (2003) Nat Rev Microbiol 1:158–164
Chen G, Ewing AG (1997) Crit Rev Neurobiol 11:59–90
Xu C-X, Yin X-F (2011) J Chromatogr A 1218:726–732
Yeung ES (1999) J Chromatogr A 830:243–262
Zabzdyr JL, Lillard SJ (2001) Trends Anal Chem 20:467–476
Wang H-Y, Bhunia AK, Lu C (2006) Biosens Bioelectron 22:582–588
Shoemaker G, Palcic M (2007) Anal Bioanal Chem 387:13–15
Křížek T, Doubnerová V, Ryšlavá H, Coufal P, Bosáková Z (2013) Anal Bioanal Chem 405:2425–2434
Sims CE, Meredith GD, Krasieva TB, Berns MW, Tromberg BJ, Allbritton NL (1998) Anal Chem 70:4570–4577
Nehme H, Nehme R, Lafite P, Routier S, Morin P (2012) Anal Chim Acta 722:127–135
Lederberg J (1950) J Bacteriol 60:381–392
Bajda M, Wieckowska A, Malawska B (2009) Acta Pol Pharm 66:357–362
Bao J, Regnier FE (1992) J Chromatogr A 608:217–224
Nehme H, Nehme R, Lafite P, Routier S, Morin P (2013) J Sep Sci 36:2151–2157
Nováková S, Van Dyck S, Van Schepdael A, Hoogmartens J, Glatz Z (2004) J Chromatogr A 1032:173–184
Zhang J, Hoogmartens J, Schepdael AV (2010) Electrophoresis 31:65–73
Paul P, Suwan J, Liu J, Dordick J, Linhardt R (2012) Anal Bioanal Chem 403:1491–1500
Husain Q (2010) Crit Rev Biotechnol 30:41–62
Yang L, Huang T, Zhu S, Zhou Y, Jiang Y, Wang S, Chen Y, Wu L, Yan X (2013) Biosens Bioelectron 48:49–55
Dimri GP, Basile G, Acosta M, Scott G, Roskelley C, Medrano EE, Linskens M, Rubelj I, Pereira-Smith O (1995) Proc Natl Acad Sci 92:9363–9367
Itahana K, Campisi J, Dimri GP (2007) Methods Mol Biol 371:21–31
Debacq-Chainiaux F, Erusalimsky JD, Campisi J, Toussaint O (2009) Nat Protoc 4:1789–1806
Shlush L, Itzkovitz S, Cohen A, Rutenberg A, Berkovitz R, Yehezkel S, Shahar H, Selig S, Skorecki K (2011) BMC Cell Biol 12:16
Kristensen HK, Lau YY, Ewing AG (1994) J Neurosci Methods 51:183–188
Troszak GD, Rubinsky B (2010) Biomed Microdevices 12:833–840
Han F, Wang Y, Sims CE, Bachman M, Chang R, Li GP, Allbritton NL (2003) Anal Chem 75:3688–3696
Kim J-S, Kwon Y-S, Sa Y-J, Kim M-J (2011) J Agric Food Chem 59:138–144
Tenu J-P, Viratelle OM, Garnier J, Yon J (1971) Eur J Biochem 20:363–370
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning - a laboratory manual. Cold Spring Harbor Laboratory Press, New York
Wallenfels K, Weil R (1972) In: Boyer PD (ed) The enzymes, 3rd edn. vol 7. Academic Press, New York
Barman TE (1969) Enzyme handbook. Springer-Verlag, New York
Johnson KA, Goody RS (2011) Biochemistry (Mosc) 50:8264–8269
Xuan X (2008) Electrophoresis 29:33–43
Landers JP (2008) Introduction to capillary electrophoresis in capillary and microchip electrophoresis and associated microtechniques. CRC, New York
Nehmé H, Nehmé R, Lafite P, Routier S, Morin P (2013) J Chromatogr A. doi:10.1016/j.chroma.2013.08.046
Shifrin S, Steers E (1967) Biochim Biophys Acta 133:454–462
Wutor VC, Togo CA, Pletschke BI (2007) Chemosphere 68:622–627
Stahl JW, Catherman AD, Sampath RK, Seneviratne CA, Strein TG (2011) Electrophoresis 32:1492–1499
Harmon BJ, Leesong I, Regnier FE (1994) Anal Chem 66:3797–3805
Nehmé R, Perrin C, Cottet H, Blanchin M-D, Fabre H (2009) Electrophoresis 30:1888–1898
Nehmé R, Perrin C (2013) Highly charged polyelectrolyte coatings to prevent adsorption during protein and peptide analysis in capillary electrophoresis of biomolecules: Methods and Protocols (Methods in Molecular Biology), Nicola Volpi, Francesca Maccari (ed) Humana Press, 984
Cohn M, Horibata K (1959) J Bacteriol 78:601–612
Miller J (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, New York
Marbach A, Bettenbrock K (2012) J Biotechnol 157:82–88
Bao N, Lu C (2008) Appl Phys Lett 92
Cheng J, Sheldon EL, Wu L, Heller MJ, O'Connell JP (1998) Anal Chem 70:2321–2326
Wang HY, Bhunia AK, Lu C (2006) Biosens Bioelectron 22:582–588
Xue Q, Yeung ES (1996) J Chromatogr B Biomed Sci Appl 677:233–240
Lee S-W, Tai Y-C (1999) Sensors Actuators A Phys 73:74–79
Lau HKF (1987) J Biochem 241:567–572
Nehmé R, Perrin C, Cottet H, Blanchin MD, Fabre H (2008) Electrophoresis 29:3013–3023
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
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
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00216-013-7332-0