Skip to main content
SpringerLink
Log in

Kinetics of bioconjugate nanoparticle label binding in a sandwich-type immunoassay

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

Abstract

Nanoparticle labels have enhanced the performance of diagnostic, screening, and other measurement applications and hold further promise for more sensitive, precise, and cost-effective assay technologies. Nevertheless, a clear view of the biomolecular interactions on the molecular level is missing. Controlling the ratio of molecular recognition over undesired nonspecific adhesion is the key to improve biosensing with nanoparticles. To improve this ratio with an aim to disallow nonspecific binding, a more detailed perspective into the kinetic differences between the cases is needed. We present the application of two novel methods to determine complex binding kinetics of bioconjugate nanoparticles, interferometry, and force spectroscopy. Force spectroscopy is an atomic force microscopy technique and optical interferometry is a direct method to monitor reaction kinetics in second-hour timescale, both having steadily increasing importance in nanomedicine. The combination is perfectly suited for this purpose, due to the high sensitivity to detect binding events and the ability to investigate biological samples under physiological conditions. We have attached a single biofunctionalized nanoparticle to the outer tip apex and studied the binding behavior of the nanoparticle in a sandwich-type immunoassay using dynamic force spectroscopy in millisecond timescale. Utilization of the two novel methods allowed characterization of binding kinetics in a time range spanning from 50 ms to 4 h. These experiments allowed detection and demonstration of differences between specific and nonspecific binding. Most importantly, nonspecific binding of a nanoparticle was reduced at contact times below 100 ms with the solid-phase surface.

A single biofunctionalized nanoparticle was attached to the outer tip apex and the binding behavior of the nanoparticle in a sandwich-type immunoassay, A) without analyte, B) with analyte and C) saturating analyte concentration, was recorded using dynamic force spectroscopy in millisecond timescale. The setting allowed measurement of the association speed of nonspecific binding.

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

Similar content being viewed by others

Abbreviations

AFM:

Atomic force microscopy

BLI:

Biolayer interferometry

FDC:

Force–distance cycle

FS:

Force spectroscopy

PDF:

Probability density function

SEM:

Scanning electron microscope

TSH:

Thyroid-stimulating hormone

References

  1. Ekins RP, Chu FW (1991) Multianalyte microspot immunoassay—microanalytical “compact disk” of the future. Clin Chem 37:1955–1967

    CAS  Google Scholar 

  2. Soukka T, Härmä H, Paukkunen J, Lövgren T (2001) Utilization of kinetically enhanced monovalent binding affinity by immunoassays based on multivalent nanoparticle-antibody bioconjugates. Anal Chem 73:2254–2260

    Article  CAS  Google Scholar 

  3. Selby C (1999) Interference in immunoassay. Ann Clin Biochem 36(Pt 6):704–721

    CAS  Google Scholar 

  4. Butler JE, Ni L, Nessler R et al (1992) The physical and functional behavior of capture antibodies adsorbed on polystyrene. J Immunol Methods 150:77–90. doi:10.1016/0022-1759(92)90066-3

    Article  CAS  Google Scholar 

  5. Soukka T, Paukkunen J, Härmä H et al (2001) Supersensitive time-resolved immunofluorometric assay of free prostate-specific antigen with nanoparticle label technology. Clin Chem 47:1269–1278

    CAS  Google Scholar 

  6. Näreoja T, Määttänen A, Peltonen J et al (2009) Impact of surface defects and denaturation of capture surface proteins on nonspecific binding in immunoassays using antibody-coated polystyrene nanoparticle labels. J Immunol Methods 347:24–30. doi:10.1016/j.jim.2009.05.010

    Article  Google Scholar 

  7. Näreoja T, Vehniäinen M, Lamminmäki U et al (2009) Study on nonspecificity of an immuoassay using Eu-doped polystyrene nanoparticle labels. J Immunol Methods 345:80–89. doi:10.1016/j.jim.2009.04.008

    Article  Google Scholar 

  8. Florin EL, Moy VT, Gaub HE (1994) Adhesion forces between individual ligand-receptor pairs. Science 264:415–417

    Article  CAS  Google Scholar 

  9. Lee GU, Chrisey LA, Colton RJ (1994) Direct measurement of the forces between complementary strands of DNA. Science 266:771–773

    Article  CAS  Google Scholar 

  10. Abdiche Y, Malashock D, Pinkerton A, Pons J (2008) Determining kinetics and affinities of protein interactions using a parallel real-time label-free biosensor, the octet. Anal Biochem 377:209–217. doi:10.1016/j.ab.2008.03.035

    Article  CAS  Google Scholar 

  11. Hahn CD, Leitner C, Weinbrenner T et al (2007) Self-assembled monolayers with latent aldehydes for protein immobilization. Bioconjug Chem 18:247–253. doi:10.1021/bc060292e

    Article  CAS  Google Scholar 

  12. Riener CK, Stroh CM, Ebner A et al (2003) Simple test system for single molecule recognition force microscopy. Anal Chim Acta 479:59–75. doi:10.1016/S0003-2670(02)01373-9

    Article  CAS  Google Scholar 

  13. Ebner A, Wildling L, Kamruzzahan ASM et al (2007) A new, simple method for linking of antibodies to atomic force microscopy tips. Bioconjug Chem 18:1176–1184. doi:10.1021/bc070030s

    Article  CAS  Google Scholar 

  14. Lyubchenko YL, Gall AA, Shlyakhtenko LS (2001) Atomic force microscopy of DNA and protein-DNA complexes using functionalized mica substrates. Methods Mol Biol 148:569–578. doi:10.1385/1-59259-208-2:569

    CAS  Google Scholar 

  15. Baumgartner, Hinterdorfer, Schindler (2000) Data analysis of interaction forces measured with the atomic force microscope. Ultramicroscopy 82:85–95

    Article  CAS  Google Scholar 

  16. Kienberger F, Ebner A, Gruber HJ, Hinterdorfer P (2006) Molecular recognition imaging and force spectroscopy of single biomolecules. Acc Chem Res 39:29–36. doi:10.1021/ar050084m

    Article  CAS  Google Scholar 

  17. Beverloo HB, van Schadewijk A, Zijlmans HJ, Tanke HJ (1992) Immunochemical detection of proteins and nucleic acids on filters using small luminescent inorganic crystals as markers. Anal Biochem 203:326–334

    Article  CAS  Google Scholar 

  18. Kuningas K, Rantanen T, Karhunen U et al (2005) Simultaneous use of time-resolved fluorescence and anti-stokes photoluminescence in a bioaffinity assay. Anal Chem 77:2826–2834. doi:10.1021/ac048186y

    Article  CAS  Google Scholar 

  19. Myszka DG (1999) Improving biosensor analysis. J Mol Recognit 12:279–284. doi:10.1002/(SICI)1099-1352(199909/10)12:5<279::AID-JMR473>3.0.CO;2-3

    Article  CAS  Google Scholar 

  20. Sulchek TA, Friddle RW, Langry K et al (2005) Dynamic force spectroscopy of parallel individual Mucin1-antibody bonds. Proc Natl Acad Sci U S A 102:16638–16643. doi:10.1073/pnas.0505208102

    Article  CAS  Google Scholar 

  21. Härmä H, Soukka T, Lövgren T (2001) Europium nanoparticles and time-resolved fluorescence for ultrasensitive detection of prostate-specific antigen. Clin Chem 47:561–568

    Google Scholar 

  22. Schwesinger F, Ros R, Strunz T et al (2000) Unbinding forces of single antibody-antigen complexes correlate with their thermal dissociation rates. Proc Natl Acad Sci U S A 97:9972–9977

    Article  CAS  Google Scholar 

  23. Ikai A, Afrin R (2003) Toward mechanical manipulations of cell membranes and membrane proteins using an atomic force microscope: an invited review. Cell Biochem Biophys 39:257–277. doi:10.1385/CBB:39:3:257

    Article  CAS  Google Scholar 

  24. Ratto TV, Langry KC, Rudd RE et al (2004) Force spectroscopy of the double-tethered concanavalin-A mannose bond. Biophys J 86:2430–2437. doi:10.1016/S0006-3495(04)74299-X

    Article  CAS  Google Scholar 

  25. Wakayama J, Sekiguchi H, Akanuma S et al (2008) Methods for reducing nonspecific interaction in antibody-antigen assay via atomic force microscopy. Anal Biochem 380:51–58. doi:10.1016/j.ab.2008.05.036

    Article  CAS  Google Scholar 

  26. Merkel R, Nassoy P, Leung A et al (1999) Energy landscapes of receptor-ligand bonds explored with dynamic force spectroscopy. Nature 397:50–53. doi:10.1038/16219

    Article  CAS  Google Scholar 

  27. Fujiki T, Tsuji A, Matsumoto S et al (2010) Generation of a human anti-tumor necrosis factor-α monoclonal antibody by in vitro immunization with a multiple antigen peptide. Biosci Biotechnol Biochem 74:1836–1840

    Article  CAS  Google Scholar 

  28. Brockmann E-C, Lamminmäki U, Saviranta P (2005) Engineering dihydropteroate synthase (DHPS) for efficient expression on M13 phage. Biochim Biophys Acta (BBA) Gen Subj 1724:146–154. doi:10.1016/j.bbagen.2005.04.012

    Article  CAS  Google Scholar 

  29. Rankl C, Kienberger F, Wildling L et al (2008) Multiple receptors involved in human rhinovirus attachment to live cells. Proc Natl Acad Sci U S A 105:17778–17783. doi:10.1073/pnas.0806451105

    Article  Google Scholar 

Download references

Acknowledgments

Guenther Hesser is thanked for recording the SEM images. The Academy of Finland under grant 110174 (T.N.) and the Austrian Research Promoting Agency (FFG-MNT-ERA.NET Project IntelliTip, grant 823980 and project VO104-08-BI, grant 819703) are acknowledged for the financial support.

Author information

Authors and Affiliations

  1. Laboratory of Biophysics, Institute of Biomedicine and Medicity Research Laboratories, University of Turku, Tykistökatu 6A, 20520, Turku, Finland

    Tuomas Näreoja, Pekka E. Hänninen & Harri Härmä

  2. Biophysics Institute, Johannes Kepler University Linz, Altenbergerstrasse 69, 4040, Linz, Austria

    Andreas Ebner, Hermann J. Gruber & Peter Hinterdorfer

  3. Center for Advanced Bioanalysis, Scharitzerstrasse 6-8, 4020, Linz, Austria

    Peter Hinterdorfer

  4. Agilent Technologies, Aubrunnerweg 11, 4040, Linz, Austria

    Ferry Kienberger

  5. Institute of Biomedical Technology and BioMediTech, University of Tampere, Biokatu 6, 33520, Tampere, Finland

    Barbara Taskinen & Vesa P. Hytönen

  6. Fimlab Laboratories, Biokatu 4, 33520, Tampere, Finland

    Vesa P. Hytönen

Authors
  1. Tuomas Näreoja
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Andreas Ebner
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hermann J. Gruber
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Barbara Taskinen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ferry Kienberger
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Pekka E. Hänninen
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Vesa P. Hytönen
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Peter Hinterdorfer
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Harri Härmä
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Tuomas Näreoja.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 348 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Näreoja, T., Ebner, A., Gruber, H.J. et al. Kinetics of bioconjugate nanoparticle label binding in a sandwich-type immunoassay. Anal Bioanal Chem 406, 493–503 (2014). https://doi.org/10.1007/s00216-013-7474-0

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-013-7474-0

Keywords

Search

Navigation