Journal of Fluorescence

, Volume 25, Issue 6, pp 1567–1575 | Cite as

A Note on the use of Steady–State Fluorescence Quenching to Quantify Nanoparticle–Protein Interactions



Steady–state fluorescence quenching is a commonly used technique to investigate the interactions between proteins and nanoparticles, providing quantitative information on binding affinity, stoichiometry and cooperativity. However, a failure to account for the limitations and pitfalls of the methodology can lead to significant errors in data analysis and interpretation. Thus, in this communication we first draw attention to a few common pitfalls in the use of fluorescence quenching to study nanoparticle–protein interactions. For example, we discuss a frequent mistake in the use of the Hill equation to determine cooperativity. We also test using both simulated and experimental data the application of a model–independent method of analysis to generate true thermodynamic nanoparticle–protein binding isotherms. This model–free approach allows for a quantitative description of the interactions independent of assumptions about the nature of the binding process [Bujalowski W, Lohman TM (1987) Biochemistry 26: 3099; Schwarz G (2000) Biophys. Chem. 86: 119].


Fluorescence quenching Spectroscopic titration Hill equation Gold nanoparticles Protein adsorption 



This work was supported by the São Paulo Research Foundation (FAPESP grant # 2013/18481-5), São Paulo, Brazil, and by the National Council for Scientific and Technological Development (CNPq grant # 476784/2013-1), Brazil.

Supplementary material

10895_2015_1665_MOESM1_ESM.docx (15 kb)
ESM 1 (DOCX 15 kb)


  1. 1.
    Treuel L, Nienhaus GU (2012) Toward a molecular understanding of nanoparticle–protein interactions. Biophys Rev 4:137–147CrossRefGoogle Scholar
  2. 2.
    Cedervall T, Lynch I, Lindman S, Berggård T, Thulin E, Nilsson H, Dawson KA, Linse S (2007) Understanding the nanoparticle–protein corona using methods to quantify exchange rates and affinities of proteins for nanoparticles. Proc Natl Acad Sci U S A 104:2050–2055PubMedCentralCrossRefPubMedGoogle Scholar
  3. 3.
    Tonga GY, Saha K, Rotello VM (2014) 25th anniversary article: interfacing nanoparticles and biology: new strategies for biomedicine. Adv Mater 26:359–370PubMedCentralCrossRefPubMedGoogle Scholar
  4. 4.
    Pd P, Pelaz B, Zhang Q, Maffre P, Nienhaus GU, Parak WJ (2014) Protein corona formation around nanoparticles – from the past to the future. Mater Horiz 1:301–313CrossRefGoogle Scholar
  5. 5.
    Xiao Q, Huang S, Su W, Li P, Ma J, Luo F, Chen J, Liu Y (2013) Systematically investigations of conformation and thermodynamics of HSA adsorbed to different sizes of CdTe quantum dots. Colloids Surf B: Biointerfaces 102:76–82CrossRefPubMedGoogle Scholar
  6. 6.
    Liang J, Cheng Y, Han H (2008) Study on the interaction between bovine serum albumin and CdTe quantum dots with spectroscopic techniques. J Mol Struct 892:116–120CrossRefGoogle Scholar
  7. 7.
    Jhonsi MA, Kathiravan A, Renganathan R (2009) Spectroscopic studies on the interaction of colloidal capped CdS nanoparticles with bovine serum albumin. Colloids Surf B: Biointerfaces 72:167–172CrossRefGoogle Scholar
  8. 8.
    Shang L, Dörlich RM, Trouillet V, Bruns M, Nienhaus GU (2012) Ultrasmall fluorescent silver nanoclusters: protein adsorption and its effects on cellular responses. Nano Res 5:531–542CrossRefGoogle Scholar
  9. 9.
    Shang L, Yang L, Seiter J, Heinle M, Brenner-Weiss G, Gerthsen D, Nienhaus GU (2014) Nanoparticles interacting with proteins and cells: a systematic study of protein surface charge effects. Adv Mater Interfaces 1:2014CrossRefGoogle Scholar
  10. 10.
    Boulos SP, Davis TA, Yang JA, Lohse SE, Alkilany AM, Holland LA, Murphy CJ (2013) Nanoparticle − protein interactions: a thermodynamic and kinetic study of the adsorption of bovine serum albumin to gold nanoparticle surfaces. Langmuir 29:14984–14996CrossRefPubMedGoogle Scholar
  11. 11.
    Yang JA, Johnson BJ, Wu S, Woods WS, George JM, Murphy CJ (2013) Study of wild-type α-synuclein binding and orientation on gold nanoparticles. Langmuir 29:4603–4615CrossRefPubMedGoogle Scholar
  12. 12.
    Lacerda SHDP, Park JJ, Meuse C, Pristinski D, Becker ML, Karim A, Douglas JF (2010) Interaction of gold nanoparticles with common human blood proteins. ACSNano 4:365–379Google Scholar
  13. 13.
    Andrews AJ, Downing G, Brown K, Park Y-J, Luger1 K (2008) A thermodynamic model for Nap1-histone interactions. J Biol Chem 283:32412–32418Google Scholar
  14. 14.
    Carpenter ML, Oliver AW, Kneale GG (2001) Analysis of DNA-protein interactions by intrinsic fluorescence. Methods Mol Biol 148:491–502PubMedGoogle Scholar
  15. 15.
    Beckett D (2011) Measurement and analysis of equilibrium binding titrations: a beginner’s guide. Methods Enzymol 488:1–16CrossRefPubMedGoogle Scholar
  16. 16.
    Lissi E, Abuin E (2011) On the evaluation of the number of binding sites in proteins from steady state fluorescence measurements. J Fluoresc 21:1831–1833CrossRefPubMedGoogle Scholar
  17. 17.
    Bujalowski W, Jezewska MJ (2014) Quantitative thermodynamic analyses of spectroscopic titration curves. J Mol Struct 1077:40–50CrossRefPubMedGoogle Scholar
  18. 18.
    Mvd W (2010) Fluorescence quenching to study protein-ligand binding: common errors. J Fluoresc 20:625–629CrossRefGoogle Scholar
  19. 19.
    Mvd W, Stella L (2011) Fluorescence quenching and ligand binding: a critical discussion of a popular methodology. J Mol Struct 998:144–150CrossRefGoogle Scholar
  20. 20.
    Stella L, Mvd W, Burrows HD, Fausto R (2014) Fluorescence spectroscopy and binding: getting it right. J Mol Struct 1077:1–3CrossRefGoogle Scholar
  21. 21.
    Grossweiner LI (2000) A note on the analysis of ligand binding by the ‘double-logarithmic’ plot. J Photochem Photobiol B: Biology 58:175–177CrossRefPubMedGoogle Scholar
  22. 22.
    Lissi E, Calderon C, Campos A (2013) Evaluation of the number of binding sites in proteins from their intrinsic fluorescence: limitations and pitfalls. Photochem Photobiol 89:1413–1416CrossRefPubMedGoogle Scholar
  23. 23.
    Stella L, Capodilupo AL, Bietti M (2008) A reassessment of the association between azulene and [60]fullerene. Possible pitfalls in the determination of binding constants through fluorescence spectroscopy. Chem Commun (39):4744–4746Google Scholar
  24. 24.
    Bujalowski W, Lohman TM (1987) A general method of analysis of ligand-macromolecule equilibria using a spectroscopic signal from the ligand to monitor binding. Application to Escherichia coli Single-Strand Binding Protein-Nucleic Acid Interactions. Biochemistry 26:3099–3106CrossRefPubMedGoogle Scholar
  25. 25.
    Bujalowski W (2006) Thermodynamic and kinetic methods of analyses of protein − nucleic acid interactions. From Simpler to More Complex Systems. Biochemistry 106:556–606Google Scholar
  26. 26.
    Schwarz G (2000) A universal thermodynamic approach to analyze biomolecular binding experiments. Biophys Chem 86:119–129CrossRefPubMedGoogle Scholar
  27. 27.
    Bisswanger H (2008) Multiple equilibria. In: Bisswanger H (ed) Enzyme kinetics: principles and methods, 2nd edn. Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, pp. 7–58CrossRefGoogle Scholar
  28. 28.
    Adair GS (1925) The hemoglobin system. The Oxygen dissociation Curve of Haemoglobin. J Biol Chem 63:529–545Google Scholar
  29. 29.
    Zhang D, Nettles CB (2015) A generalized model on the effects of nanoparticles on fluorophore fluorescence in solution. J Phys Chem C 119:7941–7948CrossRefGoogle Scholar
  30. 30.
    Credi A, Prodi L (2014) Inner filter effects and other traps in quantitative spectrofluorimetric measurements: origins and methods of correction. J Mol Struct 1077:30–39CrossRefGoogle Scholar
  31. 31.
    Shang L, Brandholt S, Stockmar F, Trouillet V, Bruns M, Nienhaus GU (2012) Effect of protein adsorption on the fluorescence of ultrasmall gold nanoclusters. Small 8:661–665CrossRefPubMedGoogle Scholar
  32. 32.
    Bunz UHF, Rotello VM (2010) Gold nanoparticle–fluorophore complexes: sensitive and discerning “noses” for biosystems sensing. Angew Chem Int Ed 39:3268–3279CrossRefGoogle Scholar
  33. 33.
    Kuzmic P (1996) Program DYNAFIT for the analysis of enzyme kinetic data: application to HIV proteinase. Anal Biochem 237:260–273CrossRefPubMedGoogle Scholar
  34. 34.
    Ackerson CJ, Powell RD, Hainfeld JF (2010) Site-specific biomolecule labeling with gold clusters. Methods Enzymol 481:195–230PubMedCentralCrossRefPubMedGoogle Scholar
  35. 35.
    Sousa AA, Morgan JT, Brown PH, Adams A, Jayasekara MPS, Zhang G, Ackerson CJ, Kruhlak MJ, Leapman RD (2012) Synthesis, characterization, and direct intracellular imaging of ultrasmall and uniform glutathione-coated gold nanoparticles. Small 8:2277–2286PubMedCentralCrossRefPubMedGoogle Scholar
  36. 36.
    You C-C, Miranda OR, Gider B, Ghosh PS, Kim I-B, Erdogan B, Krovi SA, Bunz UHF, Rotello VM (2007) Detection and identification of proteins using nanoparticle–fluorescent polymer ‘chemical nose’ sensors. Nat Nanotech 2:318–323CrossRefGoogle Scholar
  37. 37.
    Aguila A, Murray RW (2000) Monolayer-protected clusters with fluorescent dansyl. Langmuir 16:5949–5954CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of BiochemistryFederal University of São PauloSão PauloBrazil

Personalised recommendations