Biophysical Characterization of Nanoparticle-Protein Interactions by Fluorescence Quenching Titration: Limitations, Pitfalls, and Application of a Model-Free Approach for Data Analysis

  • Alioscka A. Sousa
Part of the Reviews in Fluorescence book series (RFLU)


Interactions of proteins with synthetic nanoparticles (NPs) can be investigated through a number of biophysical tools which are based on the principles of fluorescence. Among these, steady-state fluorescence quenching titration is one of the most popular techniques available. However, although not typically recognized, the multisite nature of NP-protein interactions prevents a straightforward application of the fluorescence quenching methodology. In this Chapter, some of the limitations and pitfalls of fluorescence quenching titration are therefore discussed and illustrated with simulations. It is demonstrated that commonly used analysis methods of fluorescence quench data are overly simplistic and often unsuitable for a true quantification of the interactions. Using both simulated and experimental data, it is shown how NP-protein association can be quantified instead through application of a rigorous model-independent method of analysis. Similar considerations may hold in the implementation of other biophysical methods to study NP-protein interactions.


Gold nanoparticles Nanoclusters Nanoparticle-protein interactions Fluorescence spectroscopy Fluorescence quenching Hill equation Binding isotherm 



This work was supported by the São Paulo Research Foundation (FAPESP #2013/18481-5).


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© Springer Nature Switzerland AG 2018

Authors and Affiliations

  • Alioscka A. Sousa
    • 1
  1. 1.Department of BiochemistryFederal University of São PauloSão PauloBrazil

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