Simultaneous detection of membrane protein and mRNA at single extracellular vesicle level by droplet microfluidics for cancer diagnosis
Huixian Lin, Bo Li, Jingyun Guo, Xueying Mai, Haiyang Yu, Weilun Pan, Bodeng Wu, Wei Liu, Mingzhen Zhong, Tong Liao, Ye Zhang, Bo Situ, Xiaohui Yan, Yifan Liu, Chunchen Liu, Lei Zheng
Abstract
A new droplet microfluidic assay was proposed for the simultaneous detection of membrane proteins and mRNA at the single EV level, achieving high sensitivity in EVs subpopulation analysis. By quantitative identification of multiple EVs subpopulations, this method enabling the extraction of more tumor-related information and improving cancer diagnostic performance. It shows great potential in cancer diagnosis and could be extended into the investigation of other diseases. • A versatile droplet digital analysis assay was developed for simultaneous detection of membrane protein and mRNA at single EV level. • This assay has integrated the single-molecule sensitivity of droplet microfluidics with the high specificity of antigen–antibody reactions facilitated by antibody-DNA conjugates. • The simultaneous detection of protein and mRNA at the single EV level not only addresses the challenge of EV heterogeneity but also simplifies the detection procedure enhancing the fidelity of tumor message detection. • This study proposes a promising strategy for accurate identification and analysis of specific EV subgroups through the co-detection of proteins and mRNA at the single EV level enabling the extraction of more tumor-related information and improving cancer diagnostic performance. It shows great potential in cancer diagnosis and could be extended into the investigation of other diseases. 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.