Lin Huixian, Li Bo, Guo Jingyun, Mai Xueying, Yu Haiyang, Pan Weilun, Wu Bodeng, Liu Wei, Zhong Mingzhen, Liao Tong, Zhang Ye, Situ Bo, Yan Xiaohui, Liu Yifan, Liu Chunchen, Zheng Lei
Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China; Guangdong Engineering and Technology Research Center for Rapid Diagnostic Biosensors, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China.
Breast Center, Department of General Surgery, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China.
J Adv Res. 2024 Aug 26. doi: 10.1016/j.jare.2024.08.026.
Simultaneous detection of proteins and mRNA within a single extracellular vesicle (EV) enables comprehensive analysis of specific EVs subpopulations, significantly advancing cancer diagnostics. However, developing a sensitive and user-friendly approach for simultaneously detecting multidimensional biomarkers in single EV is still challenging.
To facilitate the analysis of multidimensional biomarkers in EVs and boost its clinical application, we present a versatile droplet digital system facilitating the concurrent detection of membrane proteins and mRNA at the single EV level with high sensitivity and specificity.
The antibody-DNA conjugates were firstly prepared for EVs protein biomarkers recognition and signal transformation. Coupling with the assembled triplex droplet digital PCR system, a versatile droplet digital analysis assay for simultaneous detection of membrane protein and mRNA at a single EV level was developed.
Our new droplet digital system displayed high sensitivity and specificity. Additionally, its clinical application was validated in a breast cancer cohort. As expected, this assay has demonstrated superior performance in distinguishing breast cancer from healthy individuals and benign controls through combined detection of EVs protein and mRNA markers compared to any single kind marker detections, especially for patients with breast cancer at early stage (AUC=0.9229).
Consequently, this study proposes a promising strategy for accurately identifying and analyzing specific EV subgroups through the co-detection of proteins and mRNA at the single EV level, holding significant potential for future clinical applications.
在单个细胞外囊泡（EV）中同时检测蛋白质和mRNA能够对特定的EV亚群进行全面分析，显著推动癌症诊断的发展。然而，开发一种灵敏且用户友好的方法来同时检测单个EV中的多维生物标志物仍然具有挑战性。
为了促进对EV中多维生物标志物的分析并推动其临床应用，我们提出了一种通用的液滴数字系统，该系统能够在单个EV水平上以高灵敏度和特异性同时检测膜蛋白和mRNA。
首先制备抗体-DNA偶联物用于EV蛋白生物标志物的识别和信号转换。结合组装好的三重液滴数字PCR系统，开发了一种用于在单个EV水平上同时检测膜蛋白和mRNA的通用液滴数字分析方法。
我们的新液滴数字系统显示出高灵敏度和特异性。此外，其临床应用在一个乳腺癌队列中得到了验证。正如预期的那样，与任何单一标志物检测相比，该检测方法通过联合检测EV蛋白和mRNA标志物在区分乳腺癌与健康个体及良性对照方面表现出卓越的性能，特别是对于早期乳腺癌患者（AUC = 0.9229）。
因此，本研究提出了一种有前景的策略，通过在单个EV水平上共同检测蛋白质和mRNA来准确识别和分析特定的EV亚群，在未来临床应用中具有巨大潜力。
