Department of Biomedical Sciences, Faculty of Medicine and Health Science, Macquarie University, Sydney, NSW 2109, Australia.
School of Biomedical Engineering, University of Technology Sydney, Sydney, NSW 2007, Australia.
Int J Mol Sci. 2021 Nov 6;22(21):12014. doi: 10.3390/ijms222112014.
Exosomes belong to the class of extracellular vesicles of endocytic origin, which are regarded as a promising source of cancer biomarkers in liquid biopsy. As a result, an accurate, sensitive, and specific quantification of these nano-sized particles is of significant importance. Affinity-based approaches are recognized as the most valuable technique for exosome isolation and characterization. Indeed, Affibody biomolecules are a type of protein scaffold engineered with small size and enjoy the features of high thermal stability, affinity, and specificity. While the utilization of antibodies, aptamers, and other biologically active substances for exosome detection has been reported widely, there are no reports describing Affibody molecules' usage for exosome detection. In this study, for the first time, we have proposed a novel strategy of using Affibody functionalized microbeads (AffiBeads) for exosome detection with a high degree of efficiency. As a proof-of-concept, anti-EGFR-AffiBeads were fabricated and applied to capture and detect human lung A549 cancer cell-derived EGFR-positive exosomes using flow cytometry and fluorescent microscopy. Moreover, the capture efficiency of the AffiBeads were compared with its counterpart antibody. Our results showed that the Affibody probe had a detection limit of 15.6 ng exosomes per mL (~12 exosomes per AffiBead). The approach proposed in the current study can be used for sensitive detection of low expression level markers on tumor-derived exosomes, providing a basis for early-stage cancer diagnosis.
外泌体属于内体起源的细胞外囊泡类，被认为是液体活检中癌症生物标志物的有前途的来源。因此，准确、敏感和特异性地定量这些纳米级颗粒非常重要。基于亲和力的方法被认为是外泌体分离和表征最有价值的技术。事实上，亲和体分子是一种经过工程设计的小型蛋白质支架，具有热稳定性高、亲和力强和特异性好的特点。虽然已经广泛报道了抗体、适体和其他生物活性物质用于外泌体检测，但没有报道描述亲和体分子用于外泌体检测。在本研究中，我们首次提出了一种使用亲和体功能化微珠（AffiBeads）高效检测外泌体的新策略。作为概念验证，制备了抗 EGFR-AffiBeads，并通过流式细胞术和荧光显微镜应用于捕获和检测人肺 A549 癌细胞衍生的 EGFR 阳性外泌体。此外，还比较了 AffiBeads 的捕获效率与其抗体对照。结果表明，亲和体探针的检测限为 15.6ng/mL 外泌体（~12 个外泌体/个 AffiBead）。本研究提出的方法可用于敏感检测肿瘤来源的外泌体低表达水平标志物，为早期癌症诊断提供依据。
