Studying the Protein Corona on Nanoparticles by FCS

The nanoparticle biomolecule corona: lessons learned – challenge accepted?

“…When proteins bind to the surface of NPs, the size of the NP increases, thus changing its Brownian motion, which can be detected by DLS. 36,[141][142][143][144] Autocorrelation analysis of the fluorescence emission time traces yields a characteristic time scale of diffusion, t D , from which the hydrodynamic radius (R H ), can be calculated with sub-nanometre precision by using the Stokes-Einstein equation. Because the scattered intensity scales with the particle radius to the sixth power (R H 6 ), agglomerates, which may result from NP destabilization in biological environments, may dominate the scattered intensity.…”

“…18 DLS involves the time-dependent measurement of the intensity of visible light scattered coherently by colloidal particles. 36,[141][142][143][144] FCS is only feasible with autofluorescent or fluorescently-labelled NPs, and until now has only been used to investigate the interaction of one single protein with NPs and not with a mixture of proteins. Moreover, all the particles present in a biological sample, e.g.…”

Dissociation coefficients of protein adsorption to nanoparticles as quantitative metrics for description of the protein corona: A comparison of experimental techniques and methodological relevance

“…Diffusion coefficients can be measured via observation of NPs diffusing in and out of a test volume from the autocorrelation function of signal fluctuation. Signal of the NPs can be obtained by light scattering (DLS) or by fluorescence of the NPs (FCS) [38,39]. The resulting data set d h (c(P)) of hydrodynamic diameters determined at different protein concentrations c(P) can be fitted with the Hill model, yielding the protein concentration in which half of the NP surface is saturated with protein K' D , the maximum number N max of proteins which can be bound per NP, and the Hill coefficient n [11].…”

Engineered nanoparticles interacting with cells: size matters

“…Fluorescence correlation spectroscopy (FCS) is a sensitive technique capable of measuring the hydrodynamic diameter of freely diffusing NPs, if these are either intrinsically fluorescent or have been labeled with fluorescent dyes [9]. The FCS method is based on the analysis of the duration of brief bursts of photons from individual NPs passing through a tiny focal volume of typically 1 fL (10 –15  l), from which the NP size can be calculated via autocorrelation analysis [21,22]. As with DLS, the size information comprises both the core and the ligand layer.…”