Rupert Deborah L M, Shelke Ganesh V, Emilsson Gustav, Claudio Virginia, Block Stephan, Lässer Cecilia, Dahlin Andreas, Lötvall Jan O, Bally Marta, Zhdanov Vladimir P, Höök Fredrik
Department of Physics, Chalmers University of Technology , SE-412 96 Gothenburg, Sweden.
Krefting Research Centre, Department of Internal Medicine and Clinical Nutrition, University of Gothenburg , SE-405 30 Gothenburg, Sweden.
Anal Chem. 2016 Oct 18;88(20):9980-9988. doi: 10.1021/acs.analchem.6b01860. Epub 2016 Oct 4.
Accurate concentration determination of subpopulations of extracellular vesicles (EVs), such as exosomes, is of importance both in the context of understanding their fundamental biological role and of potentially using them as disease biomarkers. In principle, this can be achieved by measuring the rate of diffusion-limited mass uptake to a sensor surface modified with a receptor designed to only bind the subpopulation of interest. However, a significant error is introduced if the targeted EV subpopulation has a size, and thus hydrodynamic diffusion coefficient, that differs from the mean size and diffusion coefficient of the whole EV population and/or if the EVs become deformed upon binding to the surface. We here demonstrate a new approach to determine the mean size (or effective film thickness) of bound nanoparticles, in general, and EV subpopulation carrying a marker of interest, in particular. The method is based on operating surface plasmon resonance simultaneously at two wavelengths with different sensing depths and using the ratio of the corresponding responses to extract the particle size on the surface. By estimating in this way the degree of deformation of adsorbed EVs, we markedly improved their bulk concentration determination and showed that EVs carrying the exosomal marker CD63 correspond to not more than around 10% of the EV sample.
准确测定细胞外囊泡（EVs）亚群（如外泌体）的浓度，对于理解其基本生物学作用以及将其用作疾病生物标志物都具有重要意义。原则上，这可以通过测量扩散限制质量摄取到用仅与感兴趣亚群结合的受体修饰的传感器表面的速率来实现。然而，如果目标EV亚群的大小以及因此的流体动力学扩散系数与整个EV群体的平均大小和扩散系数不同，和/或如果EV在与表面结合时发生变形，则会引入显著误差。我们在此展示了一种新方法，一般用于确定结合的纳米颗粒的平均大小（或有效膜厚度），特别是携带感兴趣标记的EV亚群。该方法基于在两个具有不同传感深度的波长下同时操作表面等离子体共振，并使用相应响应的比率来提取表面上的颗粒大小。通过以这种方式估计吸附的EV的变形程度，我们显著改进了它们的总体浓度测定，并表明携带外泌体标记CD63的EV不超过EV样品的约10%。
