Rotational And Translational Diffusion Of Low Molecular Weight Nanoprobes In Ficoll Solutions

Background: Cells are typically crowded environments, affecting various properties of biomolecules such as diffusion, chemical binding, molecular structure and folding, and stability. Concentrated polymeric solutions serve as models to mimic these environments. In this study, we used ficoll, a water soluble, branched polysaccharide and we focused on probing its effect on the translational diffusion and the rotational diffusion of Alexa488 fluorophores. Here, we have a tertiary system (water, probes, polymers), introducing two lengthscales: the probe size and the mesh size of the polymer solution. It is unclear how the interplay between these two lengthscales would affect the nanoprobe translation and the rotation.Methods: We combined standard fluorescence spectroscopy, fluorescence correlation spectroscopy (FCS), and fluorescence anisotropy (FA) techniques to probe changes of the fluorescence property, the translational diffusion, and the rotational diffusion of Alexa488 fluorophores (MW approximate to 885 Da) mixed in non-fluorescent -hence "invisible"-aqueous Ficoll (MW approximate to 70 kDa) solutions. We measured changes of the emission spectrum, the lifetime, and the apparent rotational and translational diffusion coefficients of the fluorophores with systematic increase of Ficoll concentration up to 1200 mg/ml at room temperature. We also used a viscometer to measure changes of the viscosity of the ficoll solutions.Results: We found that the spectrum and the lifetime of Alexa488 appeared to be insignificantly altered by the Ficoll solutions. The measured FCS functions were readily fitted with the expression describing normal particle diffusion. Notably, however, the changes of the diffusion coefficients could not be accounted for by the corresponding changes of the bulk viscosity of the Ficoll solutions as would suggest the Stokes-Einstein relations for both diffusion coefficients. Instead, we analyzed the data with the entropic model proposed by de-Gennes and his collaborators, and fitted each set of diffusion data with a stretched exponential [exp(-ac(n))] with n being related to the quality of the solvent. For both sets the fits yielded n-value close to one, suggesting a theta-like behavior of the host Ficoll-water system. However, the a-value for translation was larger than that of rotation, indicating dissimilar local entropic effects on the rotation and translation, which was not discussed by the proposers.Conclusion: We demonstrated how FCS and FA can be applied to probe changes of the translational and rotational diffusions of a nanoprobe embedded in a polymeric solution. It appeared that the entropic model suggested by de-Gennes and his collaborators was adequate to interpret the measured FCS and FA data. Further, taken altogether the results of the analysis of the data indicated that Ficoll polymers behaved as monodisperse non-interactive nanoparticles at low concentrations but were likely to interpenetrate and entangle at high concentrations.