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Ultrasensitive detection of tumor-derived small extracellular vesicles based on nonlinear hybridization chain reaction fluorescence signal amplification and immunomagnetic separation
Analyst ( IF 3.6 ) Pub Date : 2022-03-30 , DOI: 10.1039/d2an00242f
Qianqian Kong 1 , Shasha Cheng 1 , Xinyu Hu 1 , Jia You 1 , Cuiling Zhang 1 , Yuezhong Xian 1
Analyst ( IF 3.6 ) Pub Date : 2022-03-30 , DOI: 10.1039/d2an00242f
Qianqian Kong 1 , Shasha Cheng 1 , Xinyu Hu 1 , Jia You 1 , Cuiling Zhang 1 , Yuezhong Xian 1
Affiliation
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Small extracellular vesicles (sEVs) have attracted wide attention as a promising tumor biomarker. However, sensitive and selective detection of sEVs is challenging due to the low levels of sEVs in the early stage of cancers. Herein, a novel fluorescent sensor was developed for the detection of sEVs with high sensitivity and selectivity based on nonlinear hybridization chain reaction (nHCR) signal amplification and immunomagnetic separation. Firstly, sEVs were captured and enriched by CD63 antibody conjugated magnetic beads via antibody–antigen reactions. Then, cholesterol-modified DNA probes were anchored spontaneously on lipid membranes of sEVs through efficient hydrophobic interactions between the cholesterol moiety and the phospholipid bilayer of sEVs. The simultaneous recognition of the transmembrane protein and the phospholipid bilayer structure of the sEVs could effectively eliminate interferences from free proteins. The sticky ends of the cholesterol-modified DNA probes acted as the initiator to trigger nHCR to form a hyperbranched network of DNA structure that could recruit more fluorescent signal molecules for signal amplification. Under the optimal conditions, the nHCR-based strategy showed high sensitivity for the detection of sEVs with a limit of detection of 80 particles per μL. In addition, the as-constructed method was successfully applied for the analysis of clinical samples. It provides a sensitive and selective platform for the isolation and detection of sEVs in the early diagnosis of cancers.
中文翻译：
基于非线性杂交链式反应荧光信号放大和免疫磁分离的肿瘤来源小细胞外囊泡的超灵敏检测
小细胞外囊泡（sEVs）作为一种有前途的肿瘤生物标志物引起了广泛关注。然而，由于癌症早期的 sEV 水平较低，对 sEV 进行灵敏和选择性检测具有挑战性。在此，基于非线性杂交链式反应（nHCR）信号放大和免疫磁分离，开发了一种新型荧光传感器，用于检测具有高灵敏度和选择性的sEV。首先，通过 CD63 抗体偶联磁珠捕获和富集sEV抗体-抗原反应。然后，胆固醇修饰的 DNA 探针通过胆固醇部分和 sEV 的磷脂双层之间的有效疏水相互作用自发地锚定在 sEV 的脂质膜上。同时识别sEV的跨膜蛋白和磷脂双层结构可以有效消除游离蛋白的干扰。胆固醇修饰的 DNA 探针的粘性末端作为引发剂触发 nHCR 形成 DNA 结构的超支化网络，可以招募更多的荧光信号分子进行信号放大。在最佳条件下，基于 nHCR 的策略对 sEV 的检测显示出高灵敏度，检测限为每 μL 80 个颗粒。此外，所构建的方法成功地应用于临床样本的分析。它为癌症早期诊断中sEV的分离和检测提供了一个敏感和选择性的平台。
更新日期：2022-03-30
中文翻译：
基于非线性杂交链式反应荧光信号放大和免疫磁分离的肿瘤来源小细胞外囊泡的超灵敏检测
小细胞外囊泡（sEVs）作为一种有前途的肿瘤生物标志物引起了广泛关注。然而，由于癌症早期的 sEV 水平较低，对 sEV 进行灵敏和选择性检测具有挑战性。在此，基于非线性杂交链式反应（nHCR）信号放大和免疫磁分离，开发了一种新型荧光传感器，用于检测具有高灵敏度和选择性的sEV。首先，通过 CD63 抗体偶联磁珠捕获和富集sEV抗体-抗原反应。然后，胆固醇修饰的 DNA 探针通过胆固醇部分和 sEV 的磷脂双层之间的有效疏水相互作用自发地锚定在 sEV 的脂质膜上。同时识别sEV的跨膜蛋白和磷脂双层结构可以有效消除游离蛋白的干扰。胆固醇修饰的 DNA 探针的粘性末端作为引发剂触发 nHCR 形成 DNA 结构的超支化网络，可以招募更多的荧光信号分子进行信号放大。在最佳条件下，基于 nHCR 的策略对 sEV 的检测显示出高灵敏度，检测限为每 μL 80 个颗粒。此外，所构建的方法成功地应用于临床样本的分析。它为癌症早期诊断中sEV的分离和检测提供了一个敏感和选择性的平台。
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