Skip to main content
Log in

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

  • Research Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

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

  1. Cohen D, Dickerson JA, Whitmore CD, Turner EH, Palcic MM, Hindsgaul O, Dovichi NJ (2008) Annu Rev Anal Chem 1:165–190

    Article  CAS  Google Scholar 

  2. Gary RK, Kindell SM (2005) Anal Biochem 343:329–334

    Article  CAS  Google Scholar 

  3. Herwig E, Marchetti-Deschmann M, Wenz C, Rüfer A, Allmaier G (2011) Biotechnol J 6:420–427

    Article  CAS  Google Scholar 

  4. Lidstrom ME, Meldrum DR (2003) Nat Rev Microbiol 1:158–164

    Article  CAS  Google Scholar 

  5. Chen G, Ewing AG (1997) Crit Rev Neurobiol 11:59–90

    Article  Google Scholar 

  6. Xu C-X, Yin X-F (2011) J Chromatogr A 1218:726–732

    Article  CAS  Google Scholar 

  7. Yeung ES (1999) J Chromatogr A 830:243–262

    Article  CAS  Google Scholar 

  8. Zabzdyr JL, Lillard SJ (2001) Trends Anal Chem 20:467–476

    Article  CAS  Google Scholar 

  9. Wang H-Y, Bhunia AK, Lu C (2006) Biosens Bioelectron 22:582–588

    Article  CAS  Google Scholar 

  10. Shoemaker G, Palcic M (2007) Anal Bioanal Chem 387:13–15

    Article  CAS  Google Scholar 

  11. Křížek T, Doubnerová V, Ryšlavá H, Coufal P, Bosáková Z (2013) Anal Bioanal Chem 405:2425–2434

    Article  Google Scholar 

  12. Sims CE, Meredith GD, Krasieva TB, Berns MW, Tromberg BJ, Allbritton NL (1998) Anal Chem 70:4570–4577

    Article  CAS  Google Scholar 

  13. Nehme H, Nehme R, Lafite P, Routier S, Morin P (2012) Anal Chim Acta 722:127–135

    Article  CAS  Google Scholar 

  14. Lederberg J (1950) J Bacteriol 60:381–392

    CAS  Google Scholar 

  15. Bajda M, Wieckowska A, Malawska B (2009) Acta Pol Pharm 66:357–362

    CAS  Google Scholar 

  16. Bao J, Regnier FE (1992) J Chromatogr A 608:217–224

    Article  CAS  Google Scholar 

  17. Nehme H, Nehme R, Lafite P, Routier S, Morin P (2013) J Sep Sci 36:2151–2157

    Article  CAS  Google Scholar 

  18. Nováková S, Van Dyck S, Van Schepdael A, Hoogmartens J, Glatz Z (2004) J Chromatogr A 1032:173–184

    Article  Google Scholar 

  19. Zhang J, Hoogmartens J, Schepdael AV (2010) Electrophoresis 31:65–73

    Article  Google Scholar 

  20. Paul P, Suwan J, Liu J, Dordick J, Linhardt R (2012) Anal Bioanal Chem 403:1491–1500

    Article  CAS  Google Scholar 

  21. Husain Q (2010) Crit Rev Biotechnol 30:41–62

    Article  CAS  Google Scholar 

  22. Yang L, Huang T, Zhu S, Zhou Y, Jiang Y, Wang S, Chen Y, Wu L, Yan X (2013) Biosens Bioelectron 48:49–55

    Article  CAS  Google Scholar 

  23. 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

    Article  CAS  Google Scholar 

  24. Itahana K, Campisi J, Dimri GP (2007) Methods Mol Biol 371:21–31

    Article  CAS  Google Scholar 

  25. Debacq-Chainiaux F, Erusalimsky JD, Campisi J, Toussaint O (2009) Nat Protoc 4:1789–1806

    Article  Google Scholar 

  26. Shlush L, Itzkovitz S, Cohen A, Rutenberg A, Berkovitz R, Yehezkel S, Shahar H, Selig S, Skorecki K (2011) BMC Cell Biol 12:16

    Article  CAS  Google Scholar 

  27. Kristensen HK, Lau YY, Ewing AG (1994) J Neurosci Methods 51:183–188

    Article  CAS  Google Scholar 

  28. Troszak GD, Rubinsky B (2010) Biomed Microdevices 12:833–840

    Article  Google Scholar 

  29. Han F, Wang Y, Sims CE, Bachman M, Chang R, Li GP, Allbritton NL (2003) Anal Chem 75:3688–3696

    Article  CAS  Google Scholar 

  30. Kim J-S, Kwon Y-S, Sa Y-J, Kim M-J (2011) J Agric Food Chem 59:138–144

    Article  CAS  Google Scholar 

  31. Tenu J-P, Viratelle OM, Garnier J, Yon J (1971) Eur J Biochem 20:363–370

    Article  CAS  Google Scholar 

  32. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning - a laboratory manual. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  33. Wallenfels K, Weil R (1972) In: Boyer PD (ed) The enzymes, 3rd edn. vol 7. Academic Press, New York

  34. Barman TE (1969) Enzyme handbook. Springer-Verlag, New York

    Google Scholar 

  35. Johnson KA, Goody RS (2011) Biochemistry (Mosc) 50:8264–8269

    Article  CAS  Google Scholar 

  36. Xuan X (2008) Electrophoresis 29:33–43

    Article  CAS  Google Scholar 

  37. Landers JP (2008) Introduction to capillary electrophoresis in capillary and microchip electrophoresis and associated microtechniques. CRC, New York

    Google Scholar 

  38. Nehmé H, Nehmé R, Lafite P, Routier S, Morin P (2013) J Chromatogr A. doi:10.1016/j.chroma.2013.08.046

  39. Shifrin S, Steers E (1967) Biochim Biophys Acta 133:454–462

    Article  Google Scholar 

  40. Wutor VC, Togo CA, Pletschke BI (2007) Chemosphere 68:622–627

    Article  CAS  Google Scholar 

  41. Stahl JW, Catherman AD, Sampath RK, Seneviratne CA, Strein TG (2011) Electrophoresis 32:1492–1499

    Article  CAS  Google Scholar 

  42. Harmon BJ, Leesong I, Regnier FE (1994) Anal Chem 66:3797–3805

    Article  CAS  Google Scholar 

  43. Nehmé R, Perrin C, Cottet H, Blanchin M-D, Fabre H (2009) Electrophoresis 30:1888–1898

    Article  Google Scholar 

  44. 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

  45. Cohn M, Horibata K (1959) J Bacteriol 78:601–612

    CAS  Google Scholar 

  46. Miller J (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, New York

  47. Marbach A, Bettenbrock K (2012) J Biotechnol 157:82–88

    Article  CAS  Google Scholar 

  48. Bao N, Lu C (2008) Appl Phys Lett 92

  49. Cheng J, Sheldon EL, Wu L, Heller MJ, O'Connell JP (1998) Anal Chem 70:2321–2326

    Article  CAS  Google Scholar 

  50. Wang HY, Bhunia AK, Lu C (2006) Biosens Bioelectron 22:582–588

    Article  CAS  Google Scholar 

  51. Xue Q, Yeung ES (1996) J Chromatogr B Biomed Sci Appl 677:233–240

    Article  Google Scholar 

  52. Lee S-W, Tai Y-C (1999) Sensors Actuators A Phys 73:74–79

    Article  CAS  Google Scholar 

  53. Lau HKF (1987) J Biochem 241:567–572

    CAS  Google Scholar 

  54. Nehmé R, Perrin C, Cottet H, Blanchin MD, Fabre H (2008) Electrophoresis 29:3013–3023

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Reine Nehmé.

Rights and permissions

Reprints 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

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

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

Keywords

Navigation