Skip to main content
SpringerLink
Log in

Characterisation of electrochemical immunosensor for detection of viable not-culturable forms of Legionella pneumophila in water samples

  • Original Paper
  • Published:
Chemical Papers Aims and scope Submit manuscript

Abstract

Legionella pneumophila may cause a fatal pneumonia in humans known as Legionnaires’ disease (LD). The strategies of L. pneumophila to adapt to and resist stressful environmental conditions include the ability to enter into a VBNC (viable but not culturable) state. The detection of L. pneumophila in environmental samples benefits from the use of standardised methods: for detection and enumeration following membrane filtration (AFNOR T90-431, ISO 11731) and detection and quantification by polymerase chain reaction PCR (AFNOR T90-471, ISO 12869). Culture is hampered by its inability to detect VBNC forms and PCR is unable to discriminate between live and dead bacteria. The present immunosensor was obtained by the immobilisation of a monoclonal anti-L. pneumophila antibody (MAb) on an indium-tin oxide (ITO) electrode by the self-assembled monolayers (SAMs) method using an aminosilane. The immunosensor was characterised by wettability (contact angle measurement), atomic force microscopy (AFM), confocal laser scanning microscopy (CLSM), and electrochemical impedance spectroscopy (EIS). A limit of detection of 10 bacteria per mL was observed on artificial samples.

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

Similar content being viewed by others

References

  • Ahuja, T., Rajesh Kumar, D., Kumar Tanwar, V., Sharma, V., Singh, N., & Biradar, A. M. (2010). An amperometric uric acid biosensor based on Bis[sulfosuccinimidyl] suberate crosslinker/3-aminopropyltriethoxysilane surface modified ITO glass electrode. Thin Solid Films, 519 1128–1134. DOI: 10.1016/j.tsf.2010.08.056.

    Article  CAS  Google Scholar 

  • Allegra, S., Berger, F., Berthelot, P., Grattard, F., Pozzetto, B., & Riffard, S. (2008). Use of flow cytometry to monitor Legionella viability. Applied and Environmental Microbiology, 74 7813–7816. DOI: 10.1128/aem.01364-08.

    Article  CAS  Google Scholar 

  • Allegra, S., Grattard, F., Girardot, F., Riffard, S., Pozzetto, B., & Berthelot, P. (2011a). Longitudinal evaluation of the efficacy of heat treatment procedures against Legionella spp. in hospital water systems by using a flow cytometric assay. Applied and Environmental Microbiology, 77 1268–1275. DOI: 10.1128/aem.02225-10.

    Article  CAS  Google Scholar 

  • Allegra, S., Girardot, F., Grattard, F., Berthelot, P., Helbig, J. H., Pozzetto, B., & Riffard, S. (2011b). Evaluation of an immunomagnetic separation assay in combination with cultivation to improve Legionella pneumophila serogroup 1 recovery from environmental samples. Journal of Applied Microbiology, 110 952–961. DOI: 10.1111/j.1365-2672.2011.04955.x.

    Article  CAS  Google Scholar 

  • Alleron, L., Khemiri, A., Koubar, M., Lacombe, C., Coquet, L., Cosette, P., Jouenne, T., & Frere, J. (2013). VBNC Legionella pneumophila cells are still able to produce virulence proteins. Water Research, 47 6606–6617. DOI: 10.1016/j.watres.2013.08.032.

    Article  CAS  Google Scholar 

  • Asenath-Smith, E., & Chen, W. (2008). How to prevent the loss of surface functionality derived from aminosilanes. Langmuir, 24 12405–12409. DOI: 10.1021/la802234x.

    Article  CAS  Google Scholar 

  • Braiek, M., Bekir Rokbani, K., Chrouda, A., Mrabet, B., Bakhrouf, A., Maaref, A., & Jaffrezic-Renault, N. (2012). An electrochemical immunosensor for detection of Staphylococcus aureus bacteria based on immobilization of antibodies on self-assembled monolayers-functionalized gold electrode. Biosensors, 2 417–426. DOI: 10.3390/bios2040417.

    Article  CAS  Google Scholar 

  • Benson, R. F., & Fields, B. S. (1998). Classification of the genus Legionella. Seminars in Respiratory Infections, 13 90–9.

    CAS  Google Scholar 

  • Doleans, A., Aurell, H., Reyrolle, M., Lina, G., Freney, J., Vandenesch, F., Etienne, J., & Jarraud, S. (2004). Clinical and environmental distributions of Legionella strains in France are different. Journal of Clinical Microbiology, 42 458–460. DOI: 10.1128/jcm.42.1.458-460.

    Article  Google Scholar 

  • Epalle, T., Girardot, F., Allegra, S., Maurice-Blanc, C., Garraud, O., & Riffard, S. (2015). Viable but not culturable forms of Legionella pneumophila generated after heat shock treatment are infectious for macrophage-like and alveolar epithelial cells after resuscitation on Acanthamoeba polyphaga. Microbial Ecology, 69 215–224. DOI: 10.1007/s00248-014-0470-x.

    Article  Google Scholar 

  • Escamilla-Gómez, V., Campuzano, S., Pedrero, M., & Pingarrón, J. M. (2009). Gold screen-printed-based impedimetric immunobiosensors for direct and sensitive Escherichia coli quantisation. Biosensors and Bio electronics, 24 3365–3371. DOI: 10.1016/j.bios.2009.04.047.

    Article  Google Scholar 

  • Eum, N. S., Yeom, S. H., Kwon, D. H., Kim, H. R., & Kang, S. W. (2010). Enhancement of sensitivity using gold nanorods—Antibody conjugator for detection of E. coli O157:H7. Sensors and Actuators B: Chemical, 143 784–788. DOI: 10.1016/j.snb.2009.09.054.

    Article  CAS  Google Scholar 

  • Fields, B. S., Benson, R. F., & Besser, R. E. (2002). Legionella and Legionnaires’ disease: 25 years of investigation. Clinical Microbiology Reviews, 15 506–526. DOI: 10.1128/cmr.15.3.506-526.2002.

    Article  Google Scholar 

  • Fraser, D. W., Tsai, T. R., Orenstein, W., Parkin, W. E., Beecham, H. J., Sharrar, R. G., Harris, J., Mallison, G. F., Martin, S. M., McDade, J. E., Shepard, C. C., & Brachman, P. S. (1977). Legionnaires’ disease: Description of an epidemic of pneumonia. The New England Journal of Medicine, 297 1189–1197. DOI: 10.1056/nejm197712012972201.

    Article  CAS  Google Scholar 

  • Fu, L. L., Zhang, K. W., Li, S. Q., Wang, Y. H., Huang, T. S., Zhang, A. X., & Cheng, Z. Y. (2010). In situ real-time detection of E. coli in water using antibody-coated magnetostrictive microcantilever. Sensors and Actuators B: Chemical, 150 220–225. DOI: 10.1016/j.snb.2010.07.011.

    Article  CAS  Google Scholar 

  • Gunda, N. S. K, Singh, M., Norman, L., Kaur, K., & Mitra, S. K. (2014). Optimization and characterization of biomolecule immobilization on silicon substrates using (3-aminopropyl)triethoxysilane (APTES) and glutaraldehyde linker. Applied Surface Science, 305 522–530. DOI: 10.1016/j.apsusc.2014.03.130.

    Article  CAS  Google Scholar 

  • Henke, L., & Krull, U. J. (1999). Immobilization technologies used for nucleic acid biosensors. Canadian Journal of Analytical Sciences and Spectroscopy, 44 61–70.

    CAS  Google Scholar 

  • Kim, B. R., Anderson, J. E., Mueller, S. A., Gaines, W. A., & Kendall, A. M. (2002). Literature review — efficacy of various disinfectants against Legionella in water systems. Water Research, 36 4433–4444. DOI: 10.1016/s0043-1354(02)00188-4.

    Article  CAS  Google Scholar 

  • Lei, K. F., & Leung, P. H. M. (2012). Microelectrode array biosensor for the detection of Legionella pneumophila. Microelectronic Engineering, 91 174–177. DOI: 10.1016/j.mee.2011.10.002.

    Article  CAS  Google Scholar 

  • Li, N., Brahemendra, A., Veloso, A. J., Prashar, A., Cheng, X. R., Hung, V. W. S., Guyard, C., Terebiznik, M., & Kerman, K. (2012). Disposable immunochips for the detection of Legionella pneumophila using electrochemical impedance spectroscopy. Analytical Chemistry, 84 3485–3488. DOI: 10.1021/ac3003227.

    Article  CAS  Google Scholar 

  • Mishra, S. K., Kumar, D., Biradar, A. M., & Rajesh (2012). Electrochemical impedance spectroscopy characterization of mercaptopropionic acid capped ZnS nanocrystal based bioelectrode for the detection of the cardiac biomarker—myoglobin. Bio electro chemistry, 88 118–126. DOI: 10.1016/j.bioelechem.2012.07.006.

    CAS  Google Scholar 

  • Nagare, G. D., & Mukherji, S. (2009). Characterization of silanization and antibody immobilization on spin-on glass (SOG) surface. Applied Surface Science, 255 3696–3700. DOI: 10.1016/j.apsusc.2008.10.033.

    Article  CAS  Google Scholar 

  • Oliver, J. D. (2000). The viable but nonculturable state and cellular resuscitation. In C. R. Bell, M. Brylinsky, & P. C. Johnson-Green (Eds.), Microbial biosystems: New frontiers, Proceedings of the 8th International Symposium on Microbial Ecology, August 9–14, 1998, Halifax, Canada, (pp. 723–730). Halifax, Canada: Atlantic Canada Society for Microbial Ecology.

    Google Scholar 

  • Oliver, J. D. (2005). The viable but nonculturable state in bacteria. Journal of Microbiology, 43 93–100.

    Google Scholar 

  • Puertas, S., de Gracia Villa, M., Mendoza, E., Jiménez-Jorquera, C., de la Fuente. J. M., Fernández-Sánchez, C., & Grazú, V. (2013). Improving immunosensor performance through oriented immobilization of antibodies on carbon nanotube composite surfaces. Biosensors and Bioelectronics, 43 274–280. DOI: 10.1016/j.bios.2012.12.010.

    Article  CAS  Google Scholar 

  • Qin, M., Hou, S., Wang, L. K., Feng, X. Z., Wang, R., Yang, Y. L., Wang, C., Yu, L., Shao, B., & Qiao, M. Q. (2007). Two methods for glass surface modification and their application in protein immobilization. Colloids and Surfaces B: Biointerfaces, 60 243–249. DOI: 10.1016/j.colsurfb.2007.06.018.

    Article  CAS  Google Scholar 

  • Souiri, M., Blel, N., Sboui, D., Mhamdi, L., Epalle, T., Mzoughi, R., Riffard, S., & Othmane A (2014). AFM, CLSM and EIS characterization of the immobilization of antibodies on indium-tin oxide electrode and their capture of Legionella pneumophila. Talanta, 118 224–230. DOI: 10.1016/j.talanta.2013.09.049.

    Article  CAS  Google Scholar 

  • Vashist, S. K., Dixit, C. K., MacCraith, B. D., & O’Kennedy, R. (2011). Effect of antibody immobilization strategies on the analytical performance of a surface plasmon resonance-based immunoassay. Analyst, 136 4431–4436. DOI: 10.1039/c1an15325k.

    Article  CAS  Google Scholar 

  • Vashist, S. K. (2012). Comparison of 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide based strategies to cosslink antibodies on amine-functionalized platforms for immunodiagnostic applications. Diagnostics, 2 23–33. DOI: 10.3390/diagnostics2030023.

    Article  CAS  Google Scholar 

  • WHO (2006). Gaining health: The European strategy for prevention and control of non-communicable diseases. Copenhagen, Denmark: WHO Regional Office for Europe.

    Google Scholar 

  • Zor, E., Patir, I. H., Bingol, H., & Ersoz, M. (2013). An electrochemical biosensor based on human serum albumin/graphene oxide/3-aminopropyltriethoxysilane modified ITO electrode for the enantioselective discrimination of d-and l-tryptophan. Biosensors and Bioelectronics, 42 321–325. DOI: 10.1016/j.bios.2012.10.068.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Laboratory of Avanced Materials and Interfaces, Faculty of Medicine, University of Monastir, 5019, Monastir, Tunisia

    Dejla Sboui, Mina Souiri, Manel Ben Ismail & Ali Othmane

  2. Mucosal Immunity and Pathogen Agents Group (GIMAP), EA 3064, SFR 143, Université Jean Monnet, University of Lyon, F42023, Lyon, France

    Dejla Sboui, Thibaut Epalle, Françoise Girardot, Séverine Allegra & Serge Riffard

  3. Laboratory Hubert Curien, UMR CNRS 5516, Université Jean Monnet, University of Lyon, F42023, Lyon, France

    Stephanie Reynaud

  4. Center of Confocal Microscopy (CMCM), Université Jean Monnet, University of Lyon, F42023, Lyon, France

    Sabine Palle

  5. Regional Laboratory of Hygiene, University Hospital Farhat Hached, 4000, Sousse, Tunisia

    Ridha Mzoughi

Authors
  1. Dejla Sboui
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mina Souiri
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Stephanie Reynaud
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sabine Palle
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Manel Ben Ismail
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Thibaut Epalle
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ridha Mzoughi
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Françoise Girardot
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Séverine Allegra
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Serge Riffard
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Ali Othmane
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Serge Riffard.

Additional information

S. Riffard and A. Othmane contributed equally to the work

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Sboui, D., Souiri, M., Reynaud, S. et al. Characterisation of electrochemical immunosensor for detection of viable not-culturable forms of Legionella pneumophila in water samples. Chem. Pap. 69, 1402–1410 (2015). https://doi.org/10.1515/chempap-2015-0170

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1515/chempap-2015-0170

Keywords

Search

Navigation