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Simultaneous detection of membrane protein and mRNA at single extracellular vesicle level by droplet microfluidics for cancer diagnosis
Journal of Advanced Research ( IF 11.4 ) Pub Date : 2024-08-26 , DOI: 10.1016/j.jare.2024.08.026
Huixian Lin 1 , Bo Li 1 , Jingyun Guo 2 , Xueying Mai 1 , Haiyang Yu 1 , Weilun Pan 1 , Bodeng Wu 1 , Wei Liu 1 , Mingzhen Zhong 1 , Tong Liao 1 , Ye Zhang 1 , Bo Situ 1 , Xiaohui Yan 3 , Yifan Liu 4 , Chunchen Liu 1 , Lei Zheng 1
Affiliation  

Introduction

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.

Objectives

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.

Methods

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.

Results

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).

Conclusion

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.


中文翻译:


通过液滴微流控在单个细胞外囊泡水平同时检测膜蛋白和 mRNA,用于癌症诊断


 介绍


在单个细胞外囊泡 (EV) 内同时检测蛋白质和 mRNA,能够对特定的 EV 亚群进行全面分析,从而显著推进癌症诊断。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。

 目标


为了促进 EV 中多维生物标志物的分析并促进其临床应用,我们提出了一种多功能的液滴数字系统,有助于在单个 EV 水平上同时检测膜蛋白和 mRNA,具有高灵敏度和特异性。

 方法


首先制备抗体-DNA 偶联物,用于 EVs 蛋白生物标志物识别和信号转化。与组装的三重液滴数字 PCR 系统偶联,开发了一种多功能液滴数字分析分析方法,用于在单个 EV 水平同时检测膜蛋白和 mRNA。

 结果


我们新的液滴数字系统显示出高灵敏度和特异性。此外,其临床应用在乳腺癌队列中得到了验证。正如预期的那样,与任何单一种类标志物检测相比,该检测通过联合检测 EVs 蛋白和 mRNA 标志物,在区分乳腺癌与健康个体和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)。

 结论


因此,本研究提出了一种很有前途的策略,通过在单个 EV 水平上共检测蛋白质和 mRNA 来准确识别和分析特定的 EV 亚组,具有未来临床应用的巨大潜力。
更新日期:2024-08-26
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