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Antibody-modified iron oxide nanoparticles for efficient magnetic isolation and flow cytometric determination of L. pneumophila

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Abstract

We report on the design of superparamagnetic nanoparticles capable of selectively isolating targeted bacteria (Legionella pneumophila, serogroup 1) from aqueous solutions. The surface of magnetite nanoparticles (NP) was functionalized with a heterobifunctional poly(ethylene glycol) ligand containing reactive groups for covalent coupling of polyclonal antibodies against L. pneumophila. These bioconjugates were used to label and magnetically isolate L. pneumophila. Flow cytometry revealed high separation and efficiency in this regard. The strain specificity and efficiency of the magnetic NP was tested with recombinant strains of E. coli (expressing the red fluorescent protein) and L. pneumophila (expressing the green fluorescent protein). The detection limit of the method (by flow cytometry) is 104 cells∙mL-1. The results indicate that the new multifunctional NPs are capable of selectively attracting pathogens from a complex mixture and with high efficiency. This, conceivably, paves the way to pre-concentration protocols for numerous other pathogens.

Antibody-modified iron oxide nanoparticles were developed for selective magnetic isolation of L. pneumophila bacteria from water samples. A comprehensive flow cytometry study was performed for the quantification of the bacteria.

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References

  1. Lartigue L, Innocenti C, Kalaivani T, Awwad A, Sanchez Duque MDM, Guari Y, Larionova J, Guérin C, Montero JLG, Barragan-Montero V, Arosio P, Lascialfari A, Gatteschi D, Sangregorio C (2011) Water-dispersible sugar-coated iron oxide nanoparticles. an evaluation of their relaxometric and magnetic hyperthermia properties. J Am Chem Soc 133:10459

    Article  CAS  Google Scholar 

  2. Larson TA, Bankson J, Aaron J, Sokolov K (2007) Hybrid plasmonic magnetic nanoparticles as molecular specific agents for MRI/optical imaging and photothermal therapy of cancer cells. Nanotechnology 18:325101

    Article  Google Scholar 

  3. Babes L, Denizot B, Tanguy G, Le Jeune JJ, Jallet P (1999) Synthesis of iron oxide nanoparticles used as MRI contrast agents: a parametric study. J Colloid Interface Sci 212:474

    Article  CAS  Google Scholar 

  4. Mahon E, Salvati A, Baldelli Bombelli F, Lynch I, Dawson KA (2012) Designing the nanoparticle-biomolecule interface for “targeting and therapeutic delivery”. J Control Release 161:164

    Article  CAS  Google Scholar 

  5. Kim J, Kim HS, Lee N, Kim t, Kim H, Yu T, Song IC, Moon WK, Hyeon T (2008) Multifunctional uniform nanoparticles composed of a magnetite nanocrystal core and a mesoporous silica shell for magnetic resonance and fluorescence imaging and for drug delivery. Angew Chem Int Ed Engl 47:8438

    Article  CAS  Google Scholar 

  6. Brullot W, Reddy NK, Wouters J, Valev VK, Goderis B, Vermant J, Verbiest T (2012) Versatile ferrofluids based on polyethylene glycol coated iron oxide nanoparticles. J Magn Magn Mater 324:1919

    Article  CAS  Google Scholar 

  7. Bedanta S, Kleemann W (2009) Supermagnetism. J Phys D Appl Phys 42:013001

    Article  Google Scholar 

  8. Shahbazi-Gahrouei D, Abdolahi M, Zarkesh-Esfahani SH, Laurent S, Sermeus C, Gruettner C (2013) Functionalized magnetic nanoparticles for the detection and quantitative analysis of cell surface antigen. Biomed Res Int 2013:349408

    Article  Google Scholar 

  9. Xu H, Aguilar ZP, Yang L, Kuang M, Duan H, Xiong Y, Wei H, Wang A (2011) Antibody conjugated magnetic iron oxide nanoparticles for cancer cell separation in fresh whole blood. Biomaterials 32:9758

    Article  CAS  Google Scholar 

  10. Na HB, Palui G, Rosenberg JT, Ji X, Grant SC, Mattoussi H (2012) Multidentate catechol-based polyethylene glycol oligomers provide enhanced stability and biocompatibility to iron oxide nanoparticles. ACS Nano 6:389

    Article  CAS  Google Scholar 

  11. Petri-Fink A, Steitz B, Finka A, Salaklang J, Hofmann H (2008) Effect of cell media on polymer coated superparamagnetic iron oxide nanoparticles (SPIONs): colloidal stability, cytotoxicity, and cellular uptake studies. Eur J Pharm Biopharm 68:129

    Article  CAS  Google Scholar 

  12. Ji X, Shao R, Elliott AM, Stafford RJ, Esparza-Coss E, Bankson JA, Liang G, Luo ZP, Park K, Markert JT, Li C (2007) Bifunctional gold nanoshells with a superparamagnetic iron oxide-silica core suitable for both mr imaging and photothermal therapy. J Phys Chem C Nanomater Interf 111:6245

    Article  CAS  Google Scholar 

  13. Bloemen M, Van Stappen T, Willot P, Lammertyn J, Koeckelberghs G, Geukens N, Gils A, Verbiest T (2014) Heterobifunctional PEG ligands for bioconjugation reactions on iron oxide nanoparticles. PLoS One 9:e109475

    Article  Google Scholar 

  14. Koh I, Wang X, Varughese B, Isaacs L, Ehrman SH, English DS (2006) Magnetic iron oxide nanoparticles for biorecognition: evaluation of surface coverage and activity. J Phys Chem B 110:1553

    Article  CAS  Google Scholar 

  15. Dupont D, Brullot W, Bloemen M, Verbiest T, Binnemans K (2014) Selective uptake of rare earths from aqueous solutions by EDTA-functionalized magnetic and nonmagnetic nanoparticles. ACS Appl Mater Interfaces 6:4980

    Article  CAS  Google Scholar 

  16. Horák D, Babic M, Macková H, Benes MJ (2007) Preparation and properties of magnetic nano- and microsized particles for biological and environmental separations. J Sep Sci 30:1751

    Article  Google Scholar 

  17. Chalmers JJ, Xiong Y, Jin X, Shao M, Tong X, Farag S, Zborowski M (2010) Quantification of non-specific binding of magnetic micro- and nanoparticles using cell tracking velocimetry: Implication for magnetic cell separation and detection. Biotechnol Bioeng 105:1078

    CAS  Google Scholar 

  18. Chalasani R, Vasudevan S (2012) Cyclodextrin functionalized magnetic iron oxide nanocrystals: a host-carrier for magnetic separation of non-polar molecules and arsenic from aqueous media. J Mater Chem 22:14925

    Article  CAS  Google Scholar 

  19. Atlas RM (1999) Legionella: from environmental habitats to disease pathology, detection and control. Environ Microbiol 1:283

    Article  CAS  Google Scholar 

  20. Vogel JP, Isberg RR (1999) Cell biology of Legionella pneumophila. Curr Opin Microbiol 2:30–34

    Article  CAS  Google Scholar 

  21. Behets J, Seghi F, Declerck P, Verelst L, Duvivier L, Van Damme A, Ollevier F (2003) Detection of Naegleria spp. and Naegleria fowleri: a comparison of flagellation tests, ELISA and PCR. Water Sci Technol 47:117

    CAS  Google Scholar 

  22. Behets J, Declerck P, Delaedt Y, Creemers B, Ollevier F (2006) Quantitative detection and differentiation of free-living amoeba species using SYBR green–based real-time PCR melting curve analysis. Curr Microbiol 53:506

    Article  CAS  Google Scholar 

  23. Pernin P, Pélandakis M, Rouby YA (1998) Comparative recoveries of Naegleria fowleri amoebae from seeded river water by filtration and centrifugation. Appl Environ Microbiol 64:955

    CAS  Google Scholar 

  24. Yáñez MA, Carrasco-Serrano C, Barberá VM, Catalán V (2005) Quantitative detection of Legionella pneumophila in water samples by immunomagnetic purification and real-time PCR amplification of the dotA gene. Appl Environ Microbiol 71:3433

    Article  Google Scholar 

  25. Demeke T, Jenkins GR (2010) Influence of DNA extraction methods, PCR inhibitors and quantification methods on real-time PCR assay of biotechnology-derived traits. Anal Bioanal Chem 396:1977

    Article  CAS  Google Scholar 

  26. Lundqvist M, Stigler J, Elia G, Lynch I, Verdervall T, Dawson KA (2008) Nanoparticle size and surface properties determine the protein corona with possible implications for biological impacts. Proc Natl Acad Sci U S A 105:14265

    Article  CAS  Google Scholar 

  27. Van De Craen B, Scroyen I, Vranckx C, Compernolle G, Lijnen HR, Declerck PJ, Gils A (2012) Maximal PAI-1 inhibition in vivo requires neutralizing antibodies that recognize and inhibit glycosylated PAI-1. Thromb Res 129:e126

    Article  Google Scholar 

  28. Van Stappen T, Brouwers E, Tops S, Geukens N, Vermeire S, Declerck PJ, Gils A (2014) Generation of a highly specific monoclonal anti-infliximab antibody for harmonization of TNF-coated infliximab assays. Ther Drug Monit. Just Accepted

  29. Bloemen M, Brullot W, Luong TT, Geukens N, Gils A, Verbiest T (2012) Improved functionalization of oleic acid-coated iron oxide nanoparticles for biomedical applications. J Nanoparticle Res 14:1100

    Article  Google Scholar 

  30. Köhler R, Bubert A, Goebel W, Steinert M, Hacker J, Bubert B (2000) Expression and use of the green fluorescent protein as a reporter system in Legionella pneumophila. MGG - Mol Gen Genet 262:1060

    Article  Google Scholar 

  31. Behets J, Declerck P, Delaedt Y, Creemers B, Ollevier F (2007) Development and evaluation of a Taqman duplex real-time PCR quantification method for reliable enumeration of Legionella pneumophila in water samples. J Microbiol Methods 68:137

    Article  CAS  Google Scholar 

  32. Park J, An K, Hwang Y, Park JG, Noh HJ, Kim JY, Park JH, Hwang NM, Hyeon T (2004) Ultra-large-scale syntheses of monodisperse nanocrystals. Nat Mater 3:891

    Article  CAS  Google Scholar 

  33. Schladt TD, Schneider K, Schild H, Tremel W (2011) Synthesis and bio-functionalization of magnetic nanoparticles for medical diagnosis and treatment. Dalt Trans 40:6315

    Article  CAS  Google Scholar 

  34. Hermanson G (2008) Bioconjugate techniques, 2nd ed. 1202

  35. Rao SV, Anderson KW, Bachas LG (1998) Oriented immobilization of proteins. Mikrochim Acta 128:127

    Article  CAS  Google Scholar 

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Acknowledgments

We thank prof. Johan Billen for helping with the TEM measurements. This work was financially supported by grant G.0618.11 N of the Fund for scientific research Flanders (FWO-V) and the Agency for Innovation by Science and Technology in Flanders (IWT). M. Bloemen is grateful for support from the IWT.

Conflict of interest

The authors declare that they have no conflict of interest.

Compliance with ethical standards

All legal and ethical requirements for the welfare of animals were met by the Animalium Department of the KU Leuven.

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Authors and Affiliations

  1. Department of Chemistry, KU Leuven, Celestijnenlaan 200D, box 2425, 3001, Heverlee, Belgium

    Maarten Bloemen & Thierry Verbiest

  2. Department of Biology, KU Leuven, Charles Deberiotstraat 32, Box 2439, 3000, Leuven, Belgium

    Carla Denis & Luc De Meester

  3. Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, O&N II, Herestraat 49, Box 820, 3000, Leuven, Belgium

    Miet Peeters & Ann Gils

  4. PharmAbs, The KU Leuven Antibody Center, KU Leuven, O&N II, Herestraat 49, Box 820, 3000, Leuven, Belgium

    Nick Geukens

Authors
  1. Maarten Bloemen
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  2. Carla Denis
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  4. Luc De Meester
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  7. Thierry Verbiest
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Correspondence to Maarten Bloemen.

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Bloemen, M., Denis, C., Peeters, M. et al. Antibody-modified iron oxide nanoparticles for efficient magnetic isolation and flow cytometric determination of L. pneumophila . Microchim Acta 182, 1439–1446 (2015). https://doi.org/10.1007/s00604-015-1466-z

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  • DOI: https://doi.org/10.1007/s00604-015-1466-z

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