Sydor Matthew J, Serban Monica A
BioSpectroscopy Core, Center for Biomolecular Structure and Dynamics, University of Montana, Missoula, MT 59812, USA.
Department of Biomedical and Pharmaceutical Sciences, University of Montana, Missoula, MT 59812, USA.
J Exp Theor Anal. 2023 Dec;1(2):86-96. doi: 10.3390/jeta1020007. Epub 2023 Dec 1.
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.
时间分辨荧光各向异性已被广泛用于检测与细胞及其他溶液中的粘度水平相关的双分子旋转变化。生物流体粘度的生理改变与多种病理原因有关。当前这项工作为一种测量代表生物流体的小分析物体积中粘度变化的方法提供了概念验证。本研究中使用的荧光团是荧光素二钠盐和增强型绿色荧光蛋白（EGFP）。为了评估该方法准确检测小体积样品中粘度值的能力，我们对12微升和100微升的样品进行了测量。对于两种荧光探针，所测定的粘度均未记录到随样品体积而产生的统计学显著变化。在1.02至1.31厘泊的低粘度标准液中测量了两种荧光探针的各向异性，这些标准液代表生理流体值，并且显示出随着粘度增加旋转相关时间也增加。我们还表明，可以稀释较小的流体体积以满足可用比色皿体积要求，而不会损失检测离散粘度变化的准确性。此外，通过使用含胎牛血清（FBS）的样品评估了该技术检测类似于生理流体的复杂流体中细微粘度变化的能力。分析物中FBS的存在并未改变EGFP的粘度特异性旋转相关时间，这表明该探针不与测试的分析物成分相互作用，并且能够准确反映样品粘度。我们还表明，冻融循环（反映了感兴趣的生物样品从采集到分析可能经历的温度依赖性过程）不会影响粘度测量的准确性。总体而言，我们的数据突出了使用时间分辨荧光各向异性在生物样品中进行精确粘度测量的可行性。这一发现具有重要意义，因为它可能会扩大该技术在体外诊断系统中的应用。
