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

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Book cover Reviews in Fluorescence 2017

Part of the book series: Reviews in Fluorescence ((RFLU))

Abstract

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.

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Acknowledgments

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

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Authors and Affiliations

  1. Department of Biochemistry, Federal University of São Paulo, São Paulo, Brazil

    Alioscka A. Sousa

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  1. Alioscka A. Sousa
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Editors and Affiliations

  1. Institute of Fluorescence, University of Maryland Baltimore County, Baltimore, MD, USA

    Chris D. Geddes

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Sousa, A.A. (2018). Biophysical Characterization of Nanoparticle-Protein Interactions by Fluorescence Quenching Titration: Limitations, Pitfalls, and Application of a Model-Free Approach for Data Analysis. In: Geddes, C. (eds) Reviews in Fluorescence 2017. Reviews in Fluorescence. Springer, Cham. https://doi.org/10.1007/978-3-030-01569-5_3

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