The Application of Fluorescence Anisotropy for Viscosity Measurements of Small Volume Biological Analytes

Abstract

Time-resolved fluorescence anisotropy has been extensively used to detect changes in bimolecular rotation associated with viscosity levels within cells and other solutions. Physiological alterations of the viscosity of biological fluids have been associated with numerous pathological causes. This current work serves as proof of concept for a method to measure viscosity changes in small analyte volumes representative of biological fluids. The fluorophores used in this study were fluorescein disodium salt and Enhanced Green Fluorescent Protein (EGFP). To assess the ability of the method to accurately detect viscosity values in small volume samples, we conducted measurements with 12 µL and 100 µL samples. No statistically significant changes in determined viscosities were recorded as a function of sample volume for either fluorescent probe. The anisotropy of both fluorescence probes was measured in low viscosity standards ranging from 1.02 to 1.31 cP, representative of physiological fluid values, and showed increasing rotational correlation times in response to increasing viscosity. We also showed that smaller fluid volumes can be diluted to accommodate available cuvette volume requirements without a loss in the accuracy of detecting discrete viscosity variations. Moreover, the ability of this technique to detect subtle viscosity changes in complex fluids similar to physiological ones was assessed by using fetal bovine serum (FBS) containing samples. The presence of FBS in the analytes did not alter the viscosity specific rotational correlation time of EGFP, indicating that this probe does not interact with the tested analyte components and is able to accurately reflect sample viscosity. We also showed that freeze–thaw cycles, reflective of the temperature-dependent processes that biological samples of interest could undergo from the time of collection to analyses, did not impact the viscosity measurements’ accuracy. Overall, our data highlight the feasibility of using time-resolved fluorescence anisotropy for precise viscosity measurements in biological samples. This finding is relevant as it could potentially expand the use of this technique for in vitro diagnostic systems.