Virginia G. Piper Biodesign Center for Personalized Diagnostics , Arizona State University Biodesign Institute , Tempe , Arizona 85287 , United States.
School of Biological and Health Systems Engineering , Arizona State University , Tempe , Arizona 85287 , United States.
Nano Lett. 2019 Nov 13;19(11):7623-7631. doi: 10.1021/acs.nanolett.9b02232. Epub 2019 Jul 25.
Extracellular vesicles (EVs) are of considerable interest as tumor biomarkers because tumor-derived EVs contain a broad array of information about tumor pathophysiology. However, current EV assays cannot distinguish between EV biomarker differences resulting from altered abundance of a target EV population with stable biomarker expression, altered biomarker expression in a stable target EV population, or effects arising from changes in both parameters. We now describe a rapid nanoparticle- and dye-based fluorescent immunoassay that can distinguish among these possibilities by normalizing EV biomarker levels to EV abundance. In this approach, EVs are captured from complex samples (e.g., serum), stained with a lipophilic dye, and hybridized with antibody-conjugated quantum dot probes for specific EV surface biomarkers. EV dye signal is used to quantify EV abundance and normalize EV surface biomarker expression levels. EVs from malignant and nonmalignant pancreatic cell lines exhibited similar staining, and probe-to-dye ratios did not change with EV abundance, allowing direct analysis of normalized EV biomarker expression without a separate EV quantification step. This EV biomarker normalization approach markedly improved the ability of serum levels of two pancreatic cancer biomarkers, EV EpCAM and EV EphA2, to discriminate pancreatic cancer patients from nonmalignant control subjects. The streamlined workflow and robust results of this assay are suitable for rapid translation to clinical applications and its modular design permits it to be rapidly adapted to quantitate other EV biomarkers by the simple expedient of swapping the antibody-conjugated quantum dot probes for those that recognize a different disease-specific EV biomarker.
细胞外囊泡 (EVs) 作为肿瘤标志物引起了广泛关注，因为肿瘤来源的 EVs 包含了与肿瘤病理生理学相关的广泛信息。然而，目前的 EV 检测方法无法区分由于目标 EV 群体丰度改变导致的 EV 生物标志物差异、稳定的目标 EV 群体中生物标志物表达改变，或者是由于这两个参数变化引起的差异。我们现在描述了一种快速的基于纳米颗粒和染料的荧光免疫分析方法，可以通过将 EV 生物标志物水平与 EV 丰度进行归一化来区分这些可能性。在这种方法中，EV 从复杂的样本（如血清）中被捕获，用亲脂性染料染色，并与抗体偶联的量子点探针杂交，用于特定的 EV 表面生物标志物。EV 染料信号用于定量 EV 丰度并归一化 EV 表面生物标志物表达水平。来自恶性和非恶性胰腺细胞系的 EVs 表现出相似的染色，并且探针到染料的比率不会随 EV 丰度变化而改变，从而可以直接分析归一化的 EV 生物标志物表达，而无需进行单独的 EV 定量步骤。这种 EV 生物标志物归一化方法显著提高了血清中两种胰腺癌生物标志物 EV EpCAM 和 EV EphA2 的水平，以区分胰腺癌患者和非恶性对照者的能力。该检测方法的简化工作流程和稳健的结果非常适合快速转化为临床应用，并且其模块化设计允许通过简单地更换用于识别不同疾病特异性 EV 生物标志物的抗体偶联量子点探针，快速适应定量其他 EV 生物标志物。
