Protein Immobilization on Gold Nanoparticles: Quantitative Analysis

  • Evan Decker
  • Chunsheng Bai
  • Lauren Nelless
  • Enrico Ferrari
  • Mikhail SolovievEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2118)


Conjugation of gold nanoparticles (AuNPs) with biologically relevant molecules underpins many applications in medicine and biochemistry. Immobilization of functional proteins on AuNPs often affects protein structure and function. Such effects are protein dependent and require thorough investigation using suitable quantitative tests. Good experimental design and the use of a comprehensive set of control samples are essential when characterizing the consequences of protein immobilization and its effect on protein structure and function. However, traditional approaches to making control samples, that is, immobilized protein versus protein in solution in absence of any nanoparticles, do not provide sufficiently identical reaction conditions and complicate interpretation of the results. Accurate quantification of protein conjugation to AuNPs and ensuring complete removal of unconjugated protein remain the two key challenges in such functional assays. This report describes a simple and straightforward procedure allowing for quantitative analysis of protein conjugation to AuNPs. The principles are illustrated using fluorescence and circular dichroism measurements, and can be applied to other analytical techniques or be adapted with minor modifications for use with other proteins.

Key words

Protein Gold nanoparticles Au nanoparticles AuNPs Protein A Circular dichroism Dynamic light scattering NanoDrop Protein conjugation 



We acknowledge Diamond Light Source for time on Beamline/Lab B23 under Proposal SM20209.


  1. 1.
    Laera S, Ceccone G, Rossi F et al (2011) Measuring protein structure and stability of protein–nanoparticle systems with synchrotron radiation circular dichroism. Nano Lett 11(10):4480–4484CrossRefGoogle Scholar
  2. 2.
    Tsai D-H, DelRio FW, Keene AM et al (2011) Adsorption and conformation of serum albumin protein on gold nanoparticles investigated using dimensional measurements and in situ spectroscopic methods. Langmuir 27(6):2464–2477CrossRefGoogle Scholar
  3. 3.
    Du Y, Xia L, Jo A et al (2018) Synthesis and evaluation of doxorubicin-loaded gold nanoparticles for tumor-targeted drug delivery. Bioconjug Chem 29(2):420–430CrossRefGoogle Scholar
  4. 4.
    Zhang Z, Jia J, Lai Y et al (2010) Conjugating folic acid to gold nanoparticles through glutathione for targeting and detecting cancer cells. Bioorg Med Chem 18(15):5528–5534CrossRefGoogle Scholar
  5. 5.
    Bhattacharjee TT, Castilho ML, de Oliveira IR et al (2018) FTIR study of secondary structure changes in epidermal growth factor by gold nanoparticle conjugation. Biochim Biophys Acta 1862(3):495–500CrossRefGoogle Scholar
  6. 6.
    Bailes J, Gazi S, Ivanova R, Soloviev M (2012) Effect of gold nanoparticle conjugation on the activity and stability of functional proteins. In: Soloviev M (ed) Nanoparticles in biology and medicine. Methods in molecular biology (methods and protocols), vol 906. Humana Press, Totowa, NJGoogle Scholar
  7. 7.
    Ma W, Saccardo A, Roccatano D et al (2018) Modular assembly of proteins on nanoparticles. Nat Commun 9(1):1489CrossRefGoogle Scholar
  8. 8.
    Calzolai L, Laera S, Ceccone G et al (2013) Gold nanoparticles’ blocking effect on UV-induced damage to human serum albumin. J Nanopart Res 15(1):1412CrossRefGoogle Scholar
  9. 9.
    Capomaccio R, Osório I, Ojea-Jiménez I et al (2016) Gold nanoparticles increases UV and thermal stability of human serum albumin. Biointerphases 11(4):04B310CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Evan Decker
    • 1
  • Chunsheng Bai
    • 2
  • Lauren Nelless
    • 1
  • Enrico Ferrari
    • 2
  • Mikhail Soloviev
    • 3
    Email author
  1. 1.Centre for Biomedical Sciences, School of Biological SciencesRoyal Holloway University of LondonEghamUK
  2. 2.College of Science, School of Life SciencesUniversity of Lincoln, Brayford PoolLincolnUK
  3. 3.Centre for Biomedical Sciences, Department of Biological SciencesRoyal Holloway University of LondonEghamUK

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