{"title":"An aptamer-guided fluorescence polarisation platform for extracellular vesicle liquid biopsy","authors":"Cuong Viet Pham, Rocky Chowdhury, Shweta Patel, Satendra Kumar Jaysawal, Yingchu Hou, Huo Xu, Lee Jia, Yu-mei Zhang, Xiaowei Wang, Wei Duan, Dongxi Xiang","doi":"10.1002/jev2.12502","DOIUrl":null,"url":null,"abstract":"<p>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 × 10<sup>6</sup> EVs/mL was achieved with a linear quantification range of 5.0 × 10<sup>8</sup> to 2.0 × 10<sup>10</sup> 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.</p>","PeriodicalId":15811,"journal":{"name":"Journal of Extracellular Vesicles","volume":null,"pages":null},"PeriodicalIF":15.5000,"publicationDate":"2024-09-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jev2.12502","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Extracellular Vesicles","FirstCategoryId":"3","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/jev2.12502","RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"CELL BIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
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.
要将基于细胞外囊泡(EV)的癌症生物标记物的发现转化为个性化的精准肿瘤学,就必须开发出稳健、灵敏和特异的检测方法,以便在临床实验室中应用。虽然目前已开发出多种优雅的 EV 液体活检方法,但由于对微细加工和/或精密仪器的要求较高,大多数方法仍停留在研究原型阶段。因此,本研究将开发一种简单的 DNA 配体和基于荧光偏振的均质检测方法,无需将未结合的检测配体与结合的物种分开,即可进行 EV 检测。先用一组抗体固定 EV,然后再用针对不同 EV 生物标记物的 DNA 类似物检测它们,从而实现高特异性。这种双管齐下的策略可确保在量化生物标记物阳性 EV 之前,去除输入生物流体中的大部分(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。检测限为 5.0 × 106 EVs/mL,线性定量范围为 5.0 × 108 至 2.0 × 1010 EVs/mL。在多参数分析策略的帮助下,这种由适配体引导的荧光极化测定能够根据EVs上同一组生物标记物水平的定量差异,区分来自三种不同类型实体癌细胞的EVs。鉴于该方法简单且易于在自动临床生化分析仪中实施,该测定可用于未来基于EV的连续和实时监测,以监测新的大转移或微转移的出现、癌症的进展以及在临床癌症治疗的不同阶段对治疗的反应。
期刊介绍:
The Journal of Extracellular Vesicles is an open access research publication that focuses on extracellular vesicles, including microvesicles, exosomes, ectosomes, and apoptotic bodies. It serves as the official journal of the International Society for Extracellular Vesicles and aims to facilitate the exchange of data, ideas, and information pertaining to the chemistry, biology, and applications of extracellular vesicles. The journal covers various aspects such as the cellular and molecular mechanisms of extracellular vesicles biogenesis, technological advancements in their isolation, quantification, and characterization, the role and function of extracellular vesicles in biology, stem cell-derived extracellular vesicles and their biology, as well as the application of extracellular vesicles for pharmacological, immunological, or genetic therapies.
The Journal of Extracellular Vesicles is widely recognized and indexed by numerous services, including Biological Abstracts, BIOSIS Previews, Chemical Abstracts Service (CAS), Current Contents/Life Sciences, Directory of Open Access Journals (DOAJ), Journal Citation Reports/Science Edition, Google Scholar, ProQuest Natural Science Collection, ProQuest SciTech Collection, SciTech Premium Collection, PubMed Central/PubMed, Science Citation Index Expanded, ScienceOpen, and Scopus.