Rupert Déborah L M, Lässer Cecilia, Eldh Maria, Block Stephan, Zhdanov Vladimir P, Lotvall Jan O, Bally Marta, Höök Fredrik
Department of Applied Physics, Chalmers University of Technology , SE-412 96 Gothenburg, Sweden.
Anal Chem. 2014 Jun 17;86(12):5929-36. doi: 10.1021/ac500931f. Epub 2014 Jun 5.
Exosomes are cell-secreted nanometer-sized extracellular vesicles that have been reported to play an important role in intercellular communication. They are also considered potential diagnostic markers for various health disorders, and intense investigations are presently directed toward their use as carriers in drug-delivery and gene-therapy applications. This has generated a growing need for sensitive methods capable of accurately and specifically determining the concentration of exosomes in complex biological fluids. Here, we explore the use of label-free surface-based sensing with surface plasmon resonance (SPR) read-out to determine the concentration of exosomes in solution. Human mast cell secreted exosomes carrying the tetraspanin membrane protein CD63 were analyzed by measuring their diffusion-limited binding rate to an SPR sensor surface functionalized with anti-CD63 antibodies. The concentration of suspended exosomes was determined by first converting the SPR response into the surface-bound mass. The increase in mass uptake over time was then related to the exosome concentration in solution using a formalism describing diffusion-limited binding under controlled flow conditions. The proposed quantification method is based on a calibration and control measurements performed with proteins and synthetic lipid vesicles and takes into account (i) the influence of the broad size distribution of the exosomes on the surface coverage, (ii) the fact that their size is comparable to the ∼150 nm probing depth of SPR, and (iii) possible deformation of exosomes upon adsorption. Under those considerations, the accuracy of the concentration determination was estimated to be better than ±50% and significantly improve if the exosome deformation is negligible.
外泌体是细胞分泌的纳米级细胞外囊泡，据报道在细胞间通讯中发挥重要作用。它们也被认为是各种健康疾病的潜在诊断标志物，目前正在进行深入研究，以将其用作药物递送和基因治疗应用中的载体。这就产生了对能够准确、特异性地测定复杂生物流体中外泌体浓度的灵敏方法的日益增长的需求。在此，我们探索使用基于无标记表面传感并通过表面等离子体共振（SPR）读出的方法来测定溶液中外泌体的浓度。通过测量携带四跨膜蛋白CD63的人肥大细胞分泌的外泌体与用抗CD63抗体功能化的SPR传感器表面的扩散限制结合速率，对其进行了分析。悬浮外泌体的浓度通过首先将SPR响应转换为表面结合质量来确定。然后，使用描述在受控流动条件下扩散限制结合的形式，将随时间的质量摄取增加与溶液中的外泌体浓度相关联。所提出的定量方法基于用蛋白质和合成脂质囊泡进行的校准和对照测量，并考虑了（i）外泌体广泛的尺寸分布对表面覆盖率的影响，（ii）它们的尺寸与SPR约150 nm的探测深度相当这一事实，以及（iii）外泌体吸附时可能发生的变形。在这些考虑因素下，浓度测定的准确度估计优于±50%，如果外泌体变形可忽略不计，则准确度会显著提高。
