Ran Fengying, Huang Huimin, Shang Bing, Peng Weidong, Wu Lun, Ling Kang, Xie Xiaoyu
School of Pharmacy, Health Science Center, Xi'an Jiaotong University, Xi'an, 710061, Shanxi, China.
Sinopharm Dongfeng General Hospital, Hubei University of Medicine, Shiyan, 442008, Hubei, China.
Anal Sci. 2025 Jul 17. doi: 10.1007/s44211-025-00828-3.
Extracellular vesicles (EVs) are important biomarkers for an early diagnosis of lung cancer. Herein, we proposed an ultrasensitive fluorescent sensing platform for EVs detection, which involves aptamer and streptavidin-modified magnetic nanoparticles (SA-MB) magnetic separation technology as well as T7 RNA polymerase-assisted CRISPR/Cas13a system, which can achieve target recycling signal amplification. In this detection method, biotin-modified CD63 aptamer hybridizes first with the aptamer Blocker (T7 promoter) and then binds to SA-MB. When adding EVs, the CD63 aptamer in CD63 aptamer/Blocker/SA-MB complex captures EVs causing the release of Blocker single chain. Subsequently, large amounts of ssRNAs, which are generated with the assistance of Blocker-initiated T7 RNA polymerase, were recognized by CRISPR/Cas13a and trigger its trans-cleavage report probe (F-Q). Eventually, the report probe labeled with fluorescent dye (FAM) and quench group (BHQ) at both ends was cut to produce fluorescent signal. The designed sensor combined this with a signal amplification strategy based on T7 RNA polymerase and CRISPR/Cas13a to significantly enhance the sensitivity and specificity of EVs detection. The use of magnetic separation technology eliminates interference from complex matrices and improves EVs detection efficiency, while the introduction of T7 RNA polymerase and CRISPR/Cas13a enables multiple amplifications of the sensor signals, and enhancing the accuracy and sensitivity of the method. Ultimately, the combination of multiple amplification reactions resulted in a detection limit (LOD) for EVs as low as 60 particles/mL (approximately 1 zmol/L). In addition, this detection method can specifically distinguish EVs from other confounding substances and efficiently detect plasma EVs from lung cancer and healthy individuals in actual samples. Indicating this sensing platform is a valuable tool for early lung cancer detection.
细胞外囊泡（EVs）是肺癌早期诊断的重要生物标志物。在此，我们提出了一种用于EVs检测的超灵敏荧光传感平台，该平台涉及适体和链霉亲和素修饰的磁性纳米颗粒（SA-MB）磁分离技术以及T7 RNA聚合酶辅助的CRISPR/Cas13a系统，可实现靶标循环信号放大。在这种检测方法中，生物素修饰的CD63适体首先与适体阻断剂（T7启动子）杂交，然后与SA-MB结合。加入EVs时，CD63适体/阻断剂/SA-MB复合物中的CD63适体捕获EVs，导致阻断剂单链释放。随后，在阻断剂启动的T7 RNA聚合酶的辅助下产生的大量单链RNA被CRISPR/Cas13a识别并触发其反式切割报告探针（F-Q）。最终，两端分别标记有荧光染料（FAM）和猝灭基团（BHQ）的报告探针被切割产生荧光信号。所设计的传感器将此与基于T7 RNA聚合酶和CRISPR/Cas13a的信号放大策略相结合，显著提高了EVs检测的灵敏度和特异性。磁分离技术的使用消除了复杂基质的干扰，提高了EVs检测效率，而T7 RNA聚合酶和CRISPR/Cas13a的引入使传感器信号能够多次放大，提高了方法的准确性和灵敏度。最终，多种扩增反应的结合导致EVs的检测限（LOD）低至60个颗粒/毫升（约1 zmol/L）。此外，这种检测方法可以特异性地将EVs与其他混杂物质区分开来，并能在实际样品中有效检测肺癌患者和健康个体的血浆EVs。表明该传感平台是早期肺癌检测的有价值工具。
