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Molecular Detection and Analysis of Exosomes Using Surface-Enhanced Raman Scattering Gold Nanorods and a Miniaturized Device

机译：表面增强拉曼散射金纳米棒和小型化装置的外泌体分子检测和分析

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Exosomes are a potential source of cancer biomarkers. Probing tumor-derived exosomes can offer a potential non-invasive way to diagnose cancer, assess cancer progression, and monitor treatment responses. Novel molecular methods would facilitate exosome analysis and accelerate basic and clinical exosome research.>Methods: A standard gold-coated glass microscopy slide was used to develop a miniaturized affinity-based device to capture exosomes in a target-specific manner with the assistance of low-cost 3-D printing technology. Gold nanorods coated with QSY21 Raman reporters were used as the label agent to quantitatively detect the target proteins based on surface enhanced Raman scattering spectroscopy. The expressions of several surface protein markers on exosomes from conditioned culture media of breast cancer cells and from HER2-positive breast cancer patients were quantitatively measured. The data was statistically analyzed and compared with healthy controls.>Results: A miniaturized 17 × 5 Au array device with 2-mm well size was fabricated to capture exosomes in a target-specific manner and detect the target proteins on exosomes with surface enhanced Raman scattering gold nanorods. This assay can specifically detect exosomes with a limit of detection of 2×106 exosomes/mL and analyze over 80 purified samples on a single device within 2 h. Using the assay, we have showed that exosomes derived from MDA-MB-231, MDA-MB-468, and SKBR3 breast cancer cells give distinct protein profiles compared to exosomes derived from MCF12A normal breast cells. We have also showed that exosomes in the plasma from HER2-positive breast cancer patients exhibit significantly (P ≤ 0.01) higher level of HER2 and EpCAM than those from healthy donors.>Conclusion: We have developed a simple, inexpensive, highly efficient, and portable Raman exosome assay for detection and protein profiling of exosomes. Using the assay and model exosomes from breast cancer cells, we have showed that exosomes exhibit diagnostic surface protein markers, reflecting the protein profile of their donor cells. Through proof-of-concept studies, we have identified HER2 and EpCAM biomarkers on exosomes in plasma from HER2-positive breast cancer patients, suggesting the diagnostic potential of these markers for breast cancer diagnostics. This assay would accelerate exosome research and pave a way to the development of novel cancer liquid biopsy for cancer detection and monitoring.
机译：外来体是癌症生物标志物的潜在来源。探测肿瘤来源的外泌体可以提供一种潜在的非侵入性方式来诊断癌症，评估癌症进展并监测治疗反应。新型分子方法将有助于外泌体分析并加速基础和临床外泌体研究。>方法：使用标准的镀金玻璃显微镜载玻片开发了一种基于亲和力的小型化装置，以捕获靶标特异性的外泌体低成本的3D打印技术的帮助。涂有QSY21拉曼报告基因的金纳米棒被用作标记剂，以基于表面增强拉曼散射光谱法定量检测目标蛋白。定量测量了乳腺癌细胞条件培养基和HER2阳性乳腺癌患者外泌体上几种表面蛋白标志物的表达。对数据进行统计分析，并与健康对照组进行比较。>结果：制造了孔径为2 mm的小型17×5 Au阵列设备，可以以靶标特异性方式捕获外泌体并检测靶蛋白表面增强拉曼散射金纳米棒的外泌体上的修饰。该检测方法可特异性检测外泌体，检测极限为2×10 6 外泌体/ mL，并在2小时内在单个设备上分析80多个纯化样品。使用测定法，我们已经显示，与源自MCF12A正常乳腺癌细胞的外泌体相比，源自MDA-MB-231，MDA-MB-468和SKBR3乳腺癌细胞的外泌体具有独特的蛋白质谱。我们还显示，HER2阳性乳腺癌患者血浆中的外泌体比健康捐献者的血浆中HER2和EpCAM的水平显着更高（P≤0.01）。>结论：我们已经开发出一种简单，便宜，高效且便携式的拉曼外泌体检测方法，用于外泌体的检测和蛋白质谱分析。使用来自乳腺癌细胞的测定和模型外泌体，我们已经显示出外泌体具有诊断性表面蛋白标记，反映了其供体细胞的蛋白谱。通过概念验证研究，我们已经从HER2阳性乳腺癌患者的血浆中的外泌体中鉴定了HER2和EpCAM生物标志物，表明这些标志物在乳腺癌诊断中的诊断潜力。该测定将加速外泌体研究，并为开发用于癌症检测和监测的新型癌症液体活检铺平道路。

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