将基于细胞外囊泡 (EV) 的癌症生物标志物的发现转化为个性化精准肿瘤学需要开发稳健、灵敏和特异的检测方法，并适合临床实验室采用。虽然已经开发了多种用于 EV 液体活检的优雅方法，但由于对高水平微加工和/或复杂仪器的要求，其中大多数仍作为研究原型。因此，本研究旨在开发一种简单的 DNA 适体启用和基于荧光偏振的均质测定，无需将未结合的检测配体与结合的物质分开即可进行 EV 检测。通过使用一组抗体固定 EV，然后使用针对不同 EV 生物标志物的 DNA 适体对其进行检测，可以实现高特异性。这种双管齐下的策略可确保在量化生物标志物阳性 EV 之前去除输入生物液体中的大多数（如果不是全部）非 EV 物质，包括可溶性蛋白质、蛋白质聚集体或非囊泡颗粒。线性定量范围为 5.0 × 108 至 2.0 × 1010 EVs/mL，检测限为 5.0 × 106 EVs/mL。在多参数分析策略的推动下，这种适体引导的荧光偏振测定能够根据 EV 上相同组生物标志物水平的定量差异将 EV 与三种不同类型的实体癌细胞区分开。鉴于该方法简单且易于在自动化临床生化分析仪中实施，该测定可用于未来基于 EV 的连续实时监测，以监测新的宏观或微观转移、癌症进展以及诊所癌症管理不同阶段对治疗的反应。© 2024 作者。 《Journal of Extracellular Vesicles》由 Wiley periodicals LLC 代表国际细胞外囊泡学会出版。

The translation of discoveries on extracellular vesicle (EV) based cancer biomarkers to personalised precision oncology requires the development of robust, sensitive and specific assays that are amenable to adoption in the clinical laboratory. Whilst a variety of elegant approaches for EV liquid biopsy have been developed, most of them remain as research prototypes due to the requirement of a high level of microfabrication and/or sophisticated instruments. Hence, this study is set to develop a simple DNA aptamer-enabled and fluorescence polarisation-based homogenous assay that eliminates the need to separate unbound detection ligands from the bound species for EV detection. High specificity is achieved by immobilising EVs with one set of antibodies and subsequently detecting them with a DNA aptamer targeting a distinct EV biomarker. This two-pronged strategy ensures the removal of most, if not all, non-EV substances in the input biofluids, including soluble proteins, protein aggregates or non-vesicular particles, prior to quantifying biomarker-positive EVs. A limit of detection of 5.0 × 106 EVs/mL was achieved with a linear quantification range of 5.0 × 108 to 2.0 × 1010 EVs/mL. Facilitated by a multiple parametric analysis strategy, this aptamer-guided fluorescence polarisation assay was capable of distinguishing EVs from three different types of solid cancer cells based on quantitative differences in the levels of the same sets of biomarkers on EVs. Given the simplicity of the method and its ease of implementation in automated clinical biochemistry analysers, this assay could be exploited for future EV-based continuous and real-time monitoring of the emergence of new macro- or micro-metastasis, cancer progression as well as the response to treatment throughout different stages of cancer management in the clinic.© 2024 The Author(s). Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.