Facile, generic capture and on-fiber differentiation of exosomes via confocal immunofluorescence microscopy using a capillary-channeled polymer fiber solid-phase extraction tip
Facile, generic capture and on-fiber differentiation of exosomes via confocal immunofluorescence microscopy using a capillary-channeled polymer fiber solid-phase extraction tip
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DOI：
10.1039/d2sd00007e
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发表时间：
2022-05-19
期刊：
影响因子：
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通讯作者：
Marcus, R. Kenneth
中科院分区：
文献类型：
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作者：
Jackson, Kaylan K.;Powell, Rhonda R.;Marcus, R. Kenneth
在基础生化研究和液体活检应用中，人们对分离和表征外切体(30-150 nm，细胞外小泡(EVS))的方法非常感兴趣。这是由于外体表面生物标记物的可获得性，提供了来自其起源细胞的相关生化信息。基于外显子的技术通过较小的侵入性取样(与病理学的物理提取方法相比)具有诊断应用的潜力。这项研究展示了一种简单的自旋向下尖端方法来捕获通用外切体，然后基于免疫亲和力的荧光标记来对在聚酯毛细管通道聚合物(C-CP)纤维固定相上捕获的EV进行分类。使用针对通用EV Tetraspanin蛋白(CD81)的抗体来确认纤维表面存在具有生物活性的EV。CA125蛋白的抗体，在卵巢细胞应激的情况下上调，作为癌症标记蛋白包括在内。扫描电子显微镜和共聚焦荧光显微镜直接在捕获的纤维上进行观察，以观察捕获的囊泡的形态并评估其生物活性/特性。这份报告提供了一种有效的分离、纯化、免疫标记和荧光成像手段的概念验证，用于在单一平台上评估细胞外小泡的生物标记物。这就是整体方法的新颖性所在。能够影响整个分离、免疫标记和成像过程
There is great interest in advancing methodologies for the isolation and characterization of exosomes (30-150 nm, extracellular vesicles (EVs)) for fundamental biochemical research and liquid biopsy applications. This is due to the accessibility of exosomal surface biomarkers, providing relevant biochemical information from their cells of origin. Exosome-based techniques hold potential for diagnostic applications through less invasive sampling (versus the physical extraction methods of pathology). This study demonstrates a simple spin-down tip methodology for generic exosome capture, followed by immunoaffinity-based fluorescent labeling to classify EVs captured on a polyester capillary-channeled polymer (C-CP) fiber stationary phase. An antibody to the generic EV tetraspanin protein (CD81) is employed to confirm the presence of biologically active EVs on the fiber surface. An antibody to the CA125 protein, upregulated in the case of ovarian cell stress, is included as a cancer marker protein. Scanning electron microscopy and confocal fluorescence microscopy were performed directly on the capture fibers to visualize the morphology and assess the bioactivity/identity of captured vesicles. This report provides a proof-of-concept for an efficient means of isolating, purifying, immunolabeling, and fluorescent imaging for the biomarker assessment of extracellular vesicles on a single platform. Herein lies the novelty of the overall approach. The ability to affect the entire isolation, immunolabeling, and imaging process in