Skip to main content
SpringerLink
Log in

Synthesis and characterization of enzyme-Au bioconjugates: HRP and fluorescein-labeled HRP

  • Original Articles
  • Published:
NanoBiotechnology

Abstract

Enzyme adsorption to Au nanoparticles is important for bioanalytical, catalytic, and biotechnological applications. In this manuscript, adsorption of horseradish peroxidase (HRP) and fluorescein labeled HRP (F-HRP) to colloidal Au nanoparticles is compared. Flocculation analysis indicates some differences in adsorption behavior for the labeled and unlabeled enzymes, particularly at pH 8.0, and to a lesser extent also, at pH 4.0. Zeta potential measurements were quite similar for HRP and F-HRP and leveled off at lower enzyme:Au ratios than observed by flocculation, particularly for pH 6.0 and 8.0. Direct quantification of bound F-HRP was performed by dissolution of Au nanoparticles before fluorescence measurements. We observed F-HRP binding at greater than monolayer coverage on the particles and did not reach saturation even at high solution F-HRP:Au ratios. Indirect quantification of bound unlabeled HRP based on supernatant analysis followed similar trends. More than 50% of the specific enzyme activity was retained for conjugates with a low HRP coverage. With increasing enzyme coverage, the specific activity decreased significantly; however, the total activity per particle remained nearly constant. These findings suggest HRP multilayer formation on the Au nanoparticles, with the outermost layer of HRP contributing to the major part of the conjugate enzyme activity.

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

  1. Willner I, Basnar B, Willner B. FEBS J 2007;274:302–9.

    Article  CAS  Google Scholar 

  2. Stonehuerner JG, Zhao J, O’Daly JP, Crumbliss AL, Henkens RW. Biosens Bioelectron 1992;7:421–8.

    Article  CAS  Google Scholar 

  3. Zhao J, O’Daly JP, Henkens RW, Stonehuerner J, Crumbliss AL. Biosens Bioelectron 1996;11:493–502.

    Article  CAS  Google Scholar 

  4. Phadtare S, Vinod VP, Mukopadhyay K, Kumar A, Rao M, Chaudhari RV, et al. Biotech Bioeng 2004;85:629–37.

    Article  CAS  Google Scholar 

  5. Dyal A, Loos K, Noto M, Chang SW, Spagnoli C, Shafi KVPM, et al. J Am Chem Soc 2003;125:1684–5.

    Article  CAS  Google Scholar 

  6. Caruso F, Schüler C. Langmuir 2000;16:9595–603.

    Article  CAS  Google Scholar 

  7. Jia H, Zhu G, Wang P. Biotech Bioeng 2003;84:406–14.

    Article  CAS  Google Scholar 

  8. Mandal S, Phadtare S, Sastry M. Curr Appl Phys 2005;5:118–27.

    Article  Google Scholar 

  9. Geoghegan WD, Ackerman GA. J Histochem Cytochem 1977;25:1187–200.

    CAS  Google Scholar 

  10. Gole A, Dash C, Ramakrishnan V, Sainkar SR, Mandale AB, Rao M, et al. Langmuir 2001;17:1674–9.

    Article  CAS  Google Scholar 

  11. Gole A, Dash C, Soman C, Sainkar SR, Rao M, Sastry M. Bioconj Chem 2001;12:684–90.

    Article  CAS  Google Scholar 

  12. Gole A, Vyas S, Phadtare S, Lachke A, Sastry M. Colloids Surf B 2002;25:129–38.

    Article  CAS  Google Scholar 

  13. Long MS, Keating CD. Anal Chem 2006;78:379–86.

    Article  CAS  Google Scholar 

  14. Huang Y-F, Huang C-C, Chang H-T. Langmuir 2003;19:7498–502.

    Article  CAS  Google Scholar 

  15. Abad JM, Mertens SFL, Pita M, Fernandez VM, Schiffrin DJ. J Am Chem Soc 2005;127:5689–94.

    Article  CAS  Google Scholar 

  16. Brennan JL, Hatzakis NS, Tshikhudo TR, Dirvianskyte N, Razumas V, Patkar S, et al. Bioconj Chem 2006;17:1373–5.

    Article  CAS  Google Scholar 

  17. Vertegel AA, Siegel RW, Dordick JS. Langmuir 2004;20:6800–7.

    Article  CAS  Google Scholar 

  18. Xiao Y, Ju H-X, Chen H-Y. Anal Chim Acta 1999;391:73–82.

    Article  CAS  Google Scholar 

  19. Keating CD, Kovalesky KM, Natan MJ. J Phys Chem B 1998;102:9404–13.

    Article  CAS  Google Scholar 

  20. Lundqvist M, Sethson I, Jonsson B-H. Langmuir 2004;20:10639–47.

    Article  CAS  Google Scholar 

  21. Jiang X, Jiang J, Jin Y, Wang E, Dong S. Biomacromolecules 2005;6:46–53.

    Article  CAS  Google Scholar 

  22. Grabar KC, Freeman RG, Hommer MB, Natan MJ. Anal Chem 1995;67:735–43.

    Article  CAS  Google Scholar 

  23. Frens G. Nature Phys Sci 1973;241:20–2.

    CAS  Google Scholar 

  24. Sutherland WS, Winefordner JD. J Colloid Interface Sci 1992;148:129–41.

    Article  CAS  Google Scholar 

  25. Keating CD, Musick MD, Keefe MH, Natan MJ. J Chem Ed 1999;76:949–55.

    Article  CAS  Google Scholar 

  26. Hayat MA. Colloidal gold: principles, methods, and applications, vol 1–3. San Diego: Academic; 1989.

    Google Scholar 

  27. Delincé H, Radola BJ. Eur J Biochem 1975;52:321–30.

    Article  Google Scholar 

  28. Demers LM, Mirkin CA, Mucic RC, Reynolds RA, Letsinger RL, Elghanian R, et al. Anal Chem 2000;72:5535–41.

    Article  CAS  Google Scholar 

  29. Reyes FE. Masters thesis. The Pennsylvania State University, Chemistry Department, University Park, USA; 2005.

    Google Scholar 

  30. Faulk WP, Taylor GM. Immunochemistry 1971;8:1081–3.

    Article  CAS  Google Scholar 

  31. Quinten M, Kreibig U. Surf Sci 1986;172:557–77.

    Article  CAS  Google Scholar 

  32. Mulvaney P. Langmuir 1996;12:788–800.

    Article  CAS  Google Scholar 

  33. Haes AJ, Van Duyne RP. J Am Chem Soc 2002;124:10596–604.

    Article  CAS  Google Scholar 

  34. Hutter E, Fendler JH. Adv Mater 2004;16:1685–706.

    Article  CAS  Google Scholar 

  35. Berglund GI, Carlsson GH, Smith AT, Szoe H, Henriksen A, Hajdu J. Nature 2002;417:463–8.

    Article  CAS  Google Scholar 

  36. Goodman SL, Hodges GM, Trejdosiewicz LK, Livingston DC. J Microsc 1981;123:201–13.

    CAS  Google Scholar 

  37. de Roe C, Courtoy PJ, Baudhuin P. J Histochem Cytochem 1987;35:1191–8.

    Google Scholar 

  38. Macdonald IDG, Smith WE. Langmuir 1996;12:706–13.

    Article  CAS  Google Scholar 

  39. Regnier FE. Science 1987;238:319–23.

    Article  CAS  Google Scholar 

  40. Brewer SH, Glomm WR, Johnson MC, Knag MK, Franzen S. Langmuir 2005;21:9303–7.

    Article  CAS  Google Scholar 

  41. Delincé H, Radola BJ. Biochim Biophys Acta 1970;200:404–7.

    Google Scholar 

  42. Riè-Kraut M, Ducruix A. Methods Enzymol 1997;276:23–59.

    Article  Google Scholar 

  43. Evans DE, Wennerstr:m H. In: The colloidal domain: where physics, chemistry, biology, and technology meet. 2nd ed. New York: Wiley-VCH; 1999. p. 401–42.

    Google Scholar 

  44. Horisberger M, Vauthey M. Histochemistry 1984;80:13–8.

    Article  CAS  Google Scholar 

  45. Xie H, Tkachenko AG, Glomm WR, Ryan JA, Brennaman MK, Papanikolas JM, et al. Anal Chem 2003;75:5797–805.

    Article  CAS  Google Scholar 

  46. Calabretta M, Jamison JA, Falkner JC, Liu Y, Yuhas BD, Matthews KS, et al. Nano Lett 2005;5:963–7.

    Article  CAS  Google Scholar 

  47. Dickson RB, Willingham MC, Pastan I. J Cell Biol 1981;89:29–34.

    Article  CAS  Google Scholar 

  48. Kent SP, Caldwell TC, Siegel AL. Ala J Med Sci 1979;16:22–5.

    CAS  Google Scholar 

  49. Warchol JB, Brelinska R, Herbert DC. Histochem 1982;76:567–75.

    Article  CAS  Google Scholar 

  50. Clerc M-F, Granato DA, Horisberger M. Histochemistry 1988;89:343–9.

    Article  CAS  Google Scholar 

  51. Horisberger M, Clerc M-F. Histochemistry 1985;82:219–23.

    Article  CAS  Google Scholar 

  52. Wallace JM, Dening BM, Eden KB, Stroud RM, Long JW, Rolison DR. Langmuir 2004;20:9276–81.

    Article  CAS  Google Scholar 

  53. Wallace JM, Stroud RM, Pietron JJ, Long JW, Rolison DR. J Non-Cryst Sol 2004;350:31–8.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Chemistry, Pennsylvania State University, 16802, University Park, PA, USA

    A. -S. Cans, S. L. Dean, F. E. Reyes & C. D. Keating

Authors
  1. A. -S. Cans
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. S. L. Dean
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. F. E. Reyes
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. C. D. Keating
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to C. D. Keating.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Cans, A.S., Dean, S.L., Reyes, F.E. et al. Synthesis and characterization of enzyme-Au bioconjugates: HRP and fluorescein-labeled HRP. Nanobiotechnol 3, 12–22 (2007). https://doi.org/10.1007/s12030-007-0002-6

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12030-007-0002-6

Keywords

Search

Navigation