Wu Jiacheng, Dou Quanhao, Mao Miao, Wan Xin, Wu Minhao, Hu Tony Y, Zhang Yuanqing
State Key Laboratory of Anti-Infective Drug Discovery and Development; School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, China.
Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, China.
Nat Commun. 2025 Aug 13;16(1):7498. doi: 10.1038/s41467-025-62613-0.
Extracellular vesicles (EVs) from biological fluids can provide critical information for minimally invasive diagnostics and treatment monitoring, but their nanoscale size, low biomarker abundance, and heterogeneity pose challenges. Here, we integrate rolling circle amplification with expansion microscopy (RCA-ExM) to achieve super-resolution multi-omics profiling of single EVs using conventional fluorescence microscopy. Sensitive multimodal biomarker detection is achieved by employing RCA to detect switch hairpin probe-labeled EV membrane proteins, and EV-liposome fusion to detect EV miRNAs via delivery of specific molecular beacons and a signal-amplifying enzyme circuit. Next, hydrogel-mediated expansion is employed to enlarge the fused EVs to permit single-EV detections. RCA-ExM quantitation of miRNA-21 levels in EpCAM PD-L1 plasma EVs from a clinical cohort (n = 86) successfully distinguishes cancer patients from healthy donors and differentiates 3 categories of immunotherapy efficacy. RCA-ExM therefore exhibits significant promise for more sensitive and specific diagnostics, and treatment monitoring applications.
生物体液中的细胞外囊泡（EVs）可为微创诊断和治疗监测提供关键信息，但其纳米级尺寸、低生物标志物丰度和异质性带来了挑战。在此，我们将滚环扩增与扩张显微镜（RCA-ExM）相结合，以利用传统荧光显微镜实现单个EVs的超分辨率多组学分析。通过采用RCA检测开关发夹探针标记的EV膜蛋白，以及通过递送特定分子信标和信号放大酶回路的EV-脂质体融合来检测EV miRNA，实现了灵敏的多模态生物标志物检测。接下来，采用水凝胶介导的扩张来扩大融合后的EVs，以实现单个EV的检测。对临床队列（n = 86）中EpCAM PD-L1血浆EVs中miRNA-21水平进行的RCA-ExM定量分析成功区分了癌症患者和健康供体，并区分了3类免疫治疗疗效。因此，RCA-ExM在更灵敏和特异的诊断及治疗监测应用方面展现出巨大潜力。
