Skip to main content

Advertisement

SpringerLink
Log in

Lysis of gram-positive and gram-negative bacteria by antibacterial porous polymeric monolith formed in microfluidic biochips for sample preparation

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

Abstract

Bacterial cell lysis is demonstrated using polymeric microfluidic biochips operating via a hybrid mechanical shearing/contact killing mechanism. These biochips are fabricated from a cross-linked poly(methyl methacrylate) (X-PMMA) substrate by well-controlled, high-throughput laser micromachining. The unreacted double bonds at the surface of X-PMMA provide covalent bonding for the formation of a porous polymeric monolith (PPM), thus contributing to the mechanical stability of the biochip and eliminating the need for surface treatment. The lysis efficiency of these biochips was tested for gram-positive (Enterococcus saccharolyticus and Bacillus subtilis) and gram-negative bacteria (Escherichia coli and Pseudomonas fluorescens) and confirmed by off-chip PCR without further purification. The influence of the flow rate when pumping the bacterial suspension through the PPM, and of the hydrophobic-hydrophilic balance on the cell lysis efficiency was investigated at a cell concentration of 105 CFU/mL. It was shown that the contribution of contact killing to cell lysis was more important than that of mechanical shearing in the PPM. The biochip showed better lysis efficiency than the off-chip chemical, mechanical, and thermal lysis techniques used in this work. The biochip also acts as a filter that isolates cell debris and allows PCR-amplifiable DNA to pass through. The system performs more efficient lysis for gram-negative than for gram-positive bacteria. The biochip does not require chemical/enzymatic reagents, power consumption, or complicated design and fabrication processes, which makes it an attractive on-chip lysis device that can be used in sample preparation for genetics and point-of-care diagnostics. The biochips were reused for 20 lysis cycles without any evidence of physical damage to the PPM, significant performance degradation, or DNA carryover when they were back-flushed between cycles. The biochips efficiently lysed both gram-positive and gram-negative bacteria in about 35 min per lysis and PPM regeneration cycle.

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

Access this article

Log in via an institution

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
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  1. Lee DW, Cho Y-H (2007) Continuous electrical cell lysis device using a low dc voltage for a cell transport and rupture. Sens Actuat B-Chem 124:84–89

    Article  CAS  Google Scholar 

  2. Chang DC, Chassy BM, Saunders JA, Sowers AE (1992) Guide to electroporation and electrofusion, 164–165. Academic, San Diego

    Google Scholar 

  3. Sambrook J, Russel DW (2001) Molecular cloning: a laboratory manual, 364–370. Cold Spring Harbor, New York

    Google Scholar 

  4. Lee C-Y, Lee G-B, Lin J-L, Huang F-C, Liap C-S (2004) Integrated microfluidic systems for cell lysis, mixing/pumping and DNA amplification. J Micromech Microeng 15:1215–12233

    Article  Google Scholar 

  5. Irimia D, Tompkins RG, Toner M (2004) Single-cell chemical lysis in picoliter-scale closed volumes using a microfabricated device. Anal Chem 76:6137–6143

    Article  CAS  Google Scholar 

  6. Cichova M, Proksova M, Tothova L, Santha H, Mayer V (2012) On-line cell lysis of bacteria and its spores using a microfluidic biochip. Cent Eur J Biol 7:230–240

    Article  Google Scholar 

  7. Sethu P, Anahtar M, Moldawe LL, Tompkins RG, Tone M (2001) Continuous flow microfluidic device for rapid erythrocyte lysis. Anal Chem 76:6247–6253

    Article  Google Scholar 

  8. El-Ali J, Gaudet S, Gunther A, Sorger PK, Jensen KF (2005) Cell stimulus and lysis in a microfluidic device with segmented gas-liquid flow. Anal Chem 77:3629–3636

    Article  CAS  Google Scholar 

  9. Schilling EA, Kamholz AE, Yager P (2002) Cell lysis and protein extraction in a microfluidic device with detection by a fluorogenic enzyme assay. Anal Chem 74:1798–1804

    Article  CAS  Google Scholar 

  10. Kim Y-B, Park J-H, Chang W-J, Koo Y-M, Kim EK, Kim J-H (2006) Statistical optimization of the lysis agents for gram-negative bacterial cells in a microfluidic device. Biotechnol Bioproc E 11:288–292

    Article  CAS  Google Scholar 

  11. Hall J, Felnagle E, Fries M, Spearing S, Monaco L, Steele A (2006) Evaluation of cell lysis procedures and use of a microfluidic system for an automated DNA-based cell identification in interplanetary missions. Planet Space Sci 54:1600–1611

    Article  CAS  Google Scholar 

  12. Carlo DD, Jeong K-H, Lee LP (2003) Reagentless mechanical cell lysis by nanoscale barbs in microchannels for sample preparation. Lab Chip 3:287–291

    Article  Google Scholar 

  13. Burke J. M., Smela E. (2012) A novel surface modification technique for forming porous polymer monoliths in poly(dimethylsiloxane). Biomicrofluidics 6:016506-1-016506-10

  14. Mahalanabis M, Al-Muayad H, Kulinski MD, Altman D, Klapperich CM (2009) Cell lysis and DNA extraction of gram-positive and gram-negative bacteria from whole blood in a disposable microfluidic chip. Lab Chip 9:2811–2817

    Article  CAS  Google Scholar 

  15. Lienkamp K, Madkour AE, Musante A, Nelson CF, Nusslein K, Tew GN (2008) Antimicrobial polymers prepared by ROMP with unprecedented selectivity: a molecular construction kit approach. J Am Chem Soc 130:9836–9843

    Article  CAS  Google Scholar 

  16. Tiller JC, Liao C-J, Lewis K, Klibanov AM (2001) Designing surfaces that kill bacteria on contact. J Am Chem Soc 98:5981–5985

    CAS  Google Scholar 

  17. Wan W, Yeow JT (2012) Integration of nanoparticle cell lysis and microchip PCR for one-step rapid detection of bacteria. Biomed Microdevices 14:337–346

    Article  CAS  Google Scholar 

  18. Aly Saad Aly M, Nguon O, Gauthier M, Yeow JT (2013) Antibacterial porous polymeric monolith columns with amphiphilic and polycationic character on cross-linked PMMA substrates for cell lysis applications. RSC Adv 3:24177–24184

    Article  Google Scholar 

  19. Skudas R, Thommes M, Unger KK (2011) Monolithic silicas in separation science: characterization of the pore structure of monolithic silicas. Wiley-VCH Verlag GmbH & Co 47–80

  20. Ash D. L., Page A. F. (2010) Microvolume quantification of nucleic acids in molecular diagnostics. Poster resented in 26th Clinical Virology Symposium M78: Session II.

  21. Rao A (1999) Conformation and antimicrobial activity of linear derivatives of tachyplesin lacking disulfide bonds. Arch Biochem Biophys 361:127–134

    Article  CAS  Google Scholar 

  22. Bonasera V, Alberti S, Sacchetti A (2007) Protocol for high-sensitivity/long linear-range spectrofluorimetric DNA quantification using ethidium bromide. BioTechniques 43:173–176

    Article  CAS  Google Scholar 

  23. Kubitschek HE, Freedman ML (1971) Chromosome replication and the division cycle of Escherichia coli B/r. J Bacteriol 107:95–99

    CAS  Google Scholar 

  24. Cox RA (2004) Quantitative relationships for specific growth rates and macromolecular compositions of Mycobacterium tuberculosis, Streptomyces coelicolor A3(2) and Escherichia coli B/r: an integrative theoretical approach. Microbiology 150:1413–1426

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We would like to express our gratitude to Grand Challenges Canada and Bigtec Labs for their financial support. We also thank Prof. Michael Palmer, Mr. Mohamed S. Abdel Rahman, and Mr. Eric K. Brefo-Mensah, from the Chemistry Department at the University of Waterloo, for providing access to their lab facilities and their guidance on some of the biological concepts.

Author information

Authors and Affiliations

  1. Department of Systems Design Engineering, University of Waterloo, 200 University Ave West, Waterloo, Ontario, Canada, N2L 3G1

    Mohamed Aly Saad Aly & John Yeow

  2. Department of Chemistry, University of Waterloo, 200 University Ave West, Waterloo, Ontario, Canada, N2L 3G1

    Mario Gauthier

Authors
  1. Mohamed Aly Saad Aly
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mario Gauthier
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. John Yeow
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to John Yeow.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 504 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Aly, M.A.S., Gauthier, M. & Yeow, J. Lysis of gram-positive and gram-negative bacteria by antibacterial porous polymeric monolith formed in microfluidic biochips for sample preparation. Anal Bioanal Chem 406, 5977–5987 (2014). https://doi.org/10.1007/s00216-014-8028-9

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-014-8028-9

Keywords

Search

Navigation