Determination of Exosome Concentration in Solution Using Surface Plasmon Resonance Spectroscopy
Determination of Exosome Concentration in Solution Using Surface Plasmon Resonance Spectroscopy
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DOI：
10.1021/ac500931f
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发表时间：
2014-06-17
影响因子：
7.4
通讯作者：
Hook, Fredrik
中科院分区：
文献类型：
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作者：
Rupert, Deborah L. M.;Lasser, Cecilia;Hook, Fredrik
胞外体是细胞分泌的纳米大小的胞外囊泡，已被报道在细胞间的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记物，目前针对它们在药物输送和基因治疗应用中作为载体的使用进行了密集的研究。这就产生了对能够准确和具体地确定复杂生物液中外切体浓度的灵敏方法的日益增长的需求。在这里，我们探索了利用表面等离子体共振(SPR)读出的无标记表面传感来确定溶液中外切体的浓度。人肥大细胞分泌携带Tetraspanin膜蛋白CD63的外切体，通过测量它们与抗CD63抗体功能化的SPR传感器表面的扩散限制结合率来分析。悬浮外切体的浓度是通过首先将SPR反应转化为表面结合质量来确定的。随着时间的推移，质量吸收的增加与溶液中的外切体浓度有关，该公式描述了在受控流动条件下的扩散限制结合。所提出的定量方法基于对蛋白质和合成脂泡进行的校准和对照测量，并考虑了(I)外切体的大小分布对表面覆盖率的影响，(Ii)其大小与SPR的150 nm探测深度相似，以及(Iii)外切体在吸附时可能发生的变形。在这些考虑下，浓度测定的准确度估计优于+/-50%，如果外切体变形可以忽略不计，则显著提高。
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,similar to 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.