(345f) A Fluorescence Correlation Spectroscopy Study of Hindered Probe Diffusion In Complex Media

Fluorescence Correlation Spectroscopy (FCS) has been recognized as a promising technique for evaluating the movement of molecules within complex media. Because FCS is intrinsically a number fluctuation technique, the target material is studied at low concentrations and small volumes, in contrast to other physical techniques such as small angle neutron scattering (SANS) and nuclear magnetic resonance (NMR).

Here, we present application of FCS for the study of hindered probe diffusion in polymeric solutions. This study was mainly aimed at recapitulating the intracellular environment, where diffusion of molecules is impaired predominantly by crowding and charge-mediated binding. Intracellular diffusion of molecules has long been of interest in the area of targeted drug delivery as well as for describing basic cellular processes and yet, it is still poorly understood. In particular, the combined effect of crowding and non-specific binding has rarely been addressed, partly due to the inability to decouple their individual contributions in the heterogeneous cell environment.

In this work, we developed a model system in which both molecular crowding and charge-mediated binding were addressed independently in a controlled manner. In particular, using FCS, we obtained the translational diffusion coefficient of the positively charged protein, RNase, in solutions of dextrans of various charges (binding) and concentrations (crowding). In agreement with existing data, we observed an overall 5-fold decrease in RNase diffusivity at the highest concentration of dextran, where binding accounted for 75% and crowding for 25% of the decrease. Interestingly, binding decreased RNase diffusivity by 32% even at 0.4 μM dextran. In contrast, crowding affected diffusivity only above a crowder concentration of 20 μM. Further analysis revealed that 100 μM crowder, as compared to 1 μM binder, was needed to achieve equivalent reduction in RNase diffusivity. However, the data suggested that at a higher crowder concentration (e.g., similar to that in the cell), crowding could “mask” the effect of binding.

This is one of the first studies to highlight the relative contribution of non-specific binding and crowding to hindered diffusion in complex media and thus can facilitate future understanding of molecular transport implicated in key cellular processes. However, in order to translate this study to an in-vivo setting, we would need to take into account the turbidity of the media, since it can compromise the accuracy of the FCS measurements. Therefore, we further present the impact of multiple light scattering due to background turbidity on FCS measurements.