Skip to main content
SpringerLink
Log in

Amperometric determination of live Escherichia coli using antibody-coated paramagnetic beads

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

Abstract

Detecting and enumerating fecal coliforms, especially Escherichia coli, as indicators of fecal contamination, are essential for the quality control of supplied and recreational waters. We have developed a sensitive, inexpensive, and small-volume amperometric detection method for E. coli β-galactosidase by bead-based immunoassay. The technique uses biotin-labeled capture antibodies (Ab) immobilized on paramagnetic microbeads that have been functionalized with streptavidin (bead–Ab). The bead–Ab conjugate captures E. coli from solution. The captured E. coli is incubated in Luria Bertani (LB) broth medium with the added inducer isopropyl β-D-thiogalactopyranoside (IPTG). The induced β-galactosidase converts p-aminophenyl β-D-galactopyranoside (PAPG) into p-aminophenol (PAP), which is measured by amperometry using a gold rotating disc electrode. A good linear correlation (R2=0.989) was obtained between log cfu mL−1 E. coli and the time necessary to product a specific concentration of PAP. Amperometric detection enabled determination of 2×106 cfu mL−1 E. coli within a 30 min incubation period, and the total analysis time was less than 1 h. It was also possible to determine as few as 20 cfu mL−1 E. coli under optimized conditions within 6–7 h. This process may be easily adapted as an automated portable bioanalytical device for the rapid detection of live E. coli.

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

Similar content being viewed by others

References

  1. Swaminathan B, Feng P (1994) Rapid detection of food-borne pathogenic bacteria. Ann Rev Microbiol 48:401–406

    Article  CAS  Google Scholar 

  2. Archer DL, Kvenberg JE (1985) Incidence and cost of food-borne diarrheal disease in United States. J Food Protect 48:887–894

    Google Scholar 

  3. Ford TE (1999) Microbiological safety of drinking water: United States and global perspectives. Environ Health Perspect 107:191–206

    PubMed  Google Scholar 

  4. MacKenzie WR, Hoxie NJ, Proctor ME, Gradus MS, Blair KA, Peterson DE, Addiss JJ, Fox KR, Rose JB, Davis JP (1994) A massive outbreak in Milwaukee of Cryptosporidium infection transmitted through the public water supply. N Engl J Med 331:161–167

    Article  CAS  PubMed  Google Scholar 

  5. Centers for Disease Control and Prevention (1999) Outbreak of Escherichia coli O157:H7 and Campylobacter among attendees of the Washington Country, Fair-New York. Morb Mortal Wkly Rep 48:803–805

    Google Scholar 

  6. Kondro W (2000) E. coli outbreak deaths spark judicial inquiry in Canada. Lancet 355:2058

    Article  CAS  PubMed  Google Scholar 

  7. Thomas JH, Ronkainen-Matsuno NJ, Farrell S, Halsall HB, Heineman WR (2003) Microdrop analysis of a bead-based immunoassay. Microchem J 74:267–276

    CAS  Google Scholar 

  8. Altekruse SF, Cohen ML, Sherdlow DL (1997) Emerging foodborne disease. Emerg Infect Dis 3:285–293

    CAS  PubMed  Google Scholar 

  9. Griffin PM, Tauxe RV (1991) The epidemiology of infections caused by Escherichia coli 0157:H7, other enterohemorrhagic E. coli, and the associated hemolytic uremic syndrome, Tauxe. Epidemiol Rev 13:60–98

    CAS  PubMed  Google Scholar 

  10. Jones DL (1999) Potential health risks associated with the persistence of Escherichia coli O157 in agricultural environments. Soil Use Manag 15:76–83

    Google Scholar 

  11. Willshaw GA, Thirlwell J, Jones AP, Parry S, Salmon RL, Hickey M (1994) Vero cytotoxin-producing Escherichia coli O157 in beefburgers linked to an outbreak of diarrhea, haemorrhagic colitis and haemolytic uraemic syndrome in Britain. Lett Appl Microbiol 19:304–307

    CAS  PubMed  Google Scholar 

  12. Rice EW, Allen MJ, Brender DJ, Edberg SC (1991) Assay for beta-glucuronidase in species of the genus Escherichia and its applications for drinking-water analysis. Appl Environ Microbiol 57:592–593

    CAS  PubMed  Google Scholar 

  13. American Public Health Association (1995) Standard methods for the examination of water and wastewater, 19th edn. American Public Health Association, Washington DC, Parts 9211B, 9211C, 9221B and 9222

  14. http://vm.cfsan.fda.gov/~ebam/bam-4.html

  15. Madonna AJ, Cuyk SV, Voorhees KJ (2003) Detection of Escherichia coli using immunomagnetic separation and bacteriophage amplification coupled with matrix laser desorption/ionization time-of-flight mass spectrometry. Rapid Commun Mass Spectrom 17:257–263

    Article  CAS  PubMed  Google Scholar 

  16. Geissler K, Manafi M, Amoros I, Alonso JL (2000) Quantitative determination of total coliforms and Escherichia coli in marine waters with chromogenic and fluorogenic media. J Appl Microbiol 88:280–285

    Article  CAS  PubMed  Google Scholar 

  17. Hattenberger M, Mascher F, Kalcher K, Marth E (2001) Improved method for the fluorimetric detection of β-d-galactosidase in water. Int J Hyg Environ Health 203:281–287

    CAS  PubMed  Google Scholar 

  18. Campbell GR, Prosser J, Glover A, Killham K (2001) Detection of Escherichia coli O157:H7 in soil and water using multiplex PCR. J Appl Microbiol 91:1004–1010

    Article  CAS  PubMed  Google Scholar 

  19. Sun W, Khosravi F, Albrechtsen H, Brovko LY, Griffiths MW (2002) Comparison of ATP and in vivo bioluminescence for assessing the efficiency of immunomagnetic sorbents for live Escherichia coli O157:H7 cells. J Appl Microbiol 92:1021–1027

    Article  CAS  PubMed  Google Scholar 

  20. Prescott AM, Fricker CR (1999) Use of PNA oligonucleotides for the in situ detection of Escherichia coli in water. Mol Cell Probes 13:261–268

    Article  CAS  PubMed  Google Scholar 

  21. Hurt T, Craven RD, Harvey KG, Zhang P, Price E, Fawcett NC, Evans JA (1998) Detection of E. coli O157:H7 with a quartz crystal biosensor and asymmetric PCR. In: Presented at INABIS’98—5th Internet world congress on biomedical sciences at McMaster University, Canada, December 7–16th, 1998

  22. Stender H, Broomer AJ, Oliveira K, Perry-O’Keefe H, Hyldig-Nielsen JJ, Sage A, Coull J (2001) Rapid detection, identification, and enumeration of Escherichia coli cells in municipal water by chemiluminescent in situ hybridization. Appl Environ Microbiol 67:142–147

    Article  CAS  PubMed  Google Scholar 

  23. Yam WC, Lung ML, Ng MH (1992) Evaluation and optimization of a latex agglutination assay for detection of cholera toxin and Escherichia coli heat-labile toxin. J Clin Microbiol 30:2518–2520

    CAS  PubMed  Google Scholar 

  24. Arbault P, Buecher V, Poumerol S, Sorin ML (2000) Study of ELISA method for detection of E. coli O157 in food. Prog Biotechnol 17:359–368

    CAS  Google Scholar 

  25. Yu H, Bruno JG (1996) Immunomagnetic-electrochemiluminescent detection of Escherichia coli O157 and Salmonella typhimurium in foods and environmental water samples. Appl Environ Microbiol 62:587–592

    CAS  PubMed  Google Scholar 

  26. Ho JA, Hsiu-Wen H (2003) Procedures for preparing Escherichia coli O157:H7 immunoliposome and its application in liposome immunoassay. Anal Chem 75:4330–4334

    Article  CAS  PubMed  Google Scholar 

  27. Bouvrette P, Luong JHT (1995) Development of a flow injection analysis (FIA) immunosensor for the detection of Escherichia coli. Int J Food Microbiol 27:129–137

    Article  CAS  PubMed  Google Scholar 

  28. Ruan C, Wang H, Li Y (2002) A bienzyme electrochemical biosensor coupled with immunomagnetic separation for rapid detection of Escherichia coli O157:H7 in food samples. Trans ASAE 45:249–255

    CAS  Google Scholar 

  29. Neufeld T, Schwartz-Mittelmann A, Biran D, Ron EZ, Rishpon J (2003) Combined phage typing and amperometric detection of released enzymatic activity for the specific identification and quantification of bacteria. Anal Chem 75:580–585

    Article  CAS  PubMed  Google Scholar 

  30. Wijayawardhana CA, Halsall HB, Heineman WR (1999) Micro volume rotating disk electrode (RDE) amperometric detection for a bead-based immunoassay. Anal Chim Acta 399:3–11

    Article  CAS  Google Scholar 

  31. Wijayawardhana CA, Wittstock G, Halsall HB, Heineman WR (2000) Electrochemical immunoassay with microscopic immunomagnetic bead domains and scanning electrochemical microscopy. Electroanalysis 12:640–644

    Article  CAS  Google Scholar 

  32. Purushothama S, Kradtap S, Wijayawardhana CA, Halsall HB, Heineman WR (2001) Small volume bead assay for ovalbumin with electrochemical detection. Analyst 126:337–341

    Article  CAS  PubMed  Google Scholar 

  33. Ingle JD Jr, Crouch SR (1988) Spectrochemical analysis. Prentice Hall, NJ, USA, pp 173

    Google Scholar 

  34. Perez F, Tryland I, Mascini M, Fiksdal L (2001) Rapid detection of Escherichia coli in water by a culture-based amperometric method. Anal Chim Acta 427:149–154

    Article  CAS  Google Scholar 

  35. Mittelmann AS, Ron EZ, Rishpon J (2002) Amperometric quantification of total coliforms and specific detection of Escherichia coli. Anal Chem 74:903–907

    Article  CAS  PubMed  Google Scholar 

  36. Tims TB, Lim DV (2003) Confirmation of viable E. coli O157:H7 by enrichment and PCR after rapid biosensor detection. J Microbiol Meth 55:141–147

    Article  CAS  Google Scholar 

  37. Tu SI, Patterson D, Uknalis J, Irwin P (2000) Detection of Escherichia coli O157:H7 using immunomagnetic capture and lucerin–luciferase ATP measurement. Food Res Int 33:375–380

    Article  CAS  Google Scholar 

  38. Ertl P, Wagner M, Corton E, Mikkelsen SR (2003) Rapid identification of viable Escherichia coli subspecies with an electrochemical screen-printed biosensor array. Biosens Bioelectron 18:907–916

    Article  CAS  PubMed  Google Scholar 

  39. Perez FG, Mascini M, Tothill IE, Turner APF (1998) Immunomagnetic separation with mediated flow injection analysis amperometric detection of viable Escherichia coli O157. Anal Chem 70:2380–2386

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors would like to thank Professor Brian Kinkle in the Department of Biological Sciences, University of Cincinnati, OH, USA for his counsel and for an E. coli culture. Ismail H. Boyaci would like to thank TUBITAK (The Scientific and Technical Research Council of Turkey) for a fellowship to do research at the University of Cincinnati.

Author information

Authors and Affiliations

  1. Department of Food Engineering, Hacettepe University, Ankara 06532, Turkey

    İsmail H. Boyacı

  2. Department of Chemistry, University of Cincinnati, 210172, Cincinnati, OH, 45221-0172, USA

    Zoraida P. Aguilar, Mahmud Hossain, H. Brian Halsall, Carl J. Seliskar & William R. Heineman

Authors
  1. İsmail H. Boyacı
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zoraida P. Aguilar
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mahmud Hossain
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. H. Brian Halsall
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Carl J. Seliskar
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. William R. Heineman
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to William R. Heineman.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Boyacı, İ.H., Aguilar, Z.P., Hossain, M. et al. Amperometric determination of live Escherichia coli using antibody-coated paramagnetic beads. Anal Bioanal Chem 382, 1234–1241 (2005). https://doi.org/10.1007/s00216-005-3263-8

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-005-3263-8

Keywords

Search

Navigation