Abstract
Through statistically designed experiments, lysis agents were optimized to effectively disrupt bacterial cells in a microfluidic device. Most surfactants caused the efficient lysis of Gram-positive microbes, but not of Gram-negative bacteria. A Plackett-Burman design was used to select the components that increase the efficiency of the lysis of the Gram-negative bacteriaEscherichia coli. Using this experimental design, both lysozyme and benzalkonium chloride were shown to significantly increase the cell lysis efficiency, and ATP was extracted in proportion to the lysis efficiency. Benzalkonium chloride affected the cell membrane physically, while lysozyme destroyed the cell wall, and the amount of ATP extracted increased through the synergistic interaction of these two components. The two-factor response-surface design method was used to determine the optimum concentrations of lysozyme and benzalkonium chloride, which were found to be 202 and 99 ppm, respectively. The lysis effect was further verified by microscopic observations in the microchannels. These results indicate that Gram-negative cells can be lysed efficiently in a microfluidic device, thereby allowing the rapid detection of bacterial cells using a bioluminescence-based assay of the released ATP.
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Farkade, V. D., S. Harrison, and A. B. Pandit (2005) Heat induced translocation of proteins and enzymes within the cell: An effective way to optimize the microbial cell disruption process.Biochem. Eng. J. 23: 247–257.
Chisti, Y. and M.-Y. Murray (1986) Disruption of microbial cell for intracellular products.Enzyme Microb. Technol. 8: 194–204.
Belgrader, P., M. Okuzumi, F. Pourahmadi, D. A. Borkholder, and M. A. Northrup (2000) A microfluidic cartridge to prepare spores for PCR analysis.Biosens. Bioelectron. 14: 849–852.
Kulmala, S. and J. Suomi (2003) Current status of modern analytical luminescence methods.Anal. Chim. Acta 500: 21–69.
Selan, L., F. Berlutti, C. Passariello, M. C. Thaller, and G. Renzini (1992) Reliability of a bioluminescence ATP assay for detection of bacteria.J. Clin. Microbiol. 30: 1739–1742.
Venkateswaran, K., N. Hattori, M. T. La Duc, and R. Kern (2003) ATP as a biomarker of viable microorganisms in clean-room facilities.J. Microbiol. Methods 52: 367–377.
Thore, A., A. Lundin, and S. Ansehn (1983) Firefly luciferase ATP assay as a screening method for bacteriuria.J. Clin. Microbiol. 17: 218–224.
Thore, A., A. A. Lundin, and S. Bergman (1975) Detection of bacteriuria by luciferase assay of adenosine triphosphate.J. Clin. Microbiol. 1: 1–8.
Lai-King, N. G., D. E. Taylor, and M. E. Stiles (1985) Estimation ofCamphylobacter spp. in broth culture by bioluminescence assay of ATP.Appl. Environ. Microbiol. 49: 730–731.
Blum, L. J. (1997) Bio- and chemiluminescent sensors. pp. 37–68. World Scientific, Singapore.
Brock, T. D. (2002)Biology of Microbiology. pp. 56–57. Prentice Hall, Englewood Cliffs, NJ, USA.
Miller, T. E. (1969) Killing and lysis of gram-negative bacteria through the synergistic effect of hydrogen peroxide, ascorbic acid, and lysozyme.J. Bacteriol. 98: 949–955.
Kim, S. K., B. S. Lee, J. G. Lee, H. J. Seo, and E. K. Kim (2003) Continuous water toxicity monitoring using immobilizedPhotobacterium phosphoreum.Biotechnol. Bioprocess Eng. 8: 147–150.
Effenhauser, C. S., G. J. M. Bruin, A. Paulus, and M. Ehrat (1997) Integrated capillary electrophoresis on flexible silicone microdevices: Analysis of DNA restriction fragments and detection of single DNA molecules on microchips.Anal. Chem. 69: 3451–3457.
Duffy, D. C., J. C. McDonald, O. J. A. Schueller, and G. M. Whitesides (1998) Rapid prototyping of microfluidic systems in poly(dimethylsiloxane).Anal. Chem. 70: 4974–4984.
Kim, H., H.-J. Eom, J. Lee, J. Han, and N. S. Han (2004) Statistical optimization of medium composition for growth ofLeuconostoc citreum.Biotechnol. Bioprocess Eng. 9: 278–284.
Yucel, N. and H. Ulusoy (2006) A Turkey survey of hygiene indicator bacteria andYersinia enterocolitica in raw milk and cheese samples.Food Control 17: 383–388.
Ho, J. (2002) Future of biological aerosol detection.Anal. Chim. Acta 457: 125–148.
L'Hostis, E., P. E. Michel, G. C. Fiaccabrino, D. J. Strike, N. F. de Rooij, and M. Koudelka-Hep (2000) Microreactor and electrochemical detectors fabricated using Si and EPON SU-8.Sens. Actuators B Chem. 64: 156–162.
Roda, A., P. Pasini, M. Mirasoli, E. Michelini, and M. Guardigli (2004) Biotechnological applications of bioluminescence and chemiluminescence.Trends Biotechnol. 22: 295–303.
Li, C., W.-C. Lee, and K. H. Lee (2003) Affinity separations using microfabricated microfluidic devices:In situ photopolymerization and use in protein separations.Biotechnol. Bioprocess Eng. 8: 240–245.
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An erratum to this article is available at http://dx.doi.org/10.1007/BF02932084.
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Kim, YB., Park, JH., Chang, WJ. et al. Statistical optimization of the lysis agents for Gram-negative bacterial cells in a microfluidic device. Biotechnol. Bioprocess Eng. 11, 288–292 (2006). https://doi.org/10.1007/BF03026242
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DOI: https://doi.org/10.1007/BF03026242