Simultaneous detection of membrane protein and mRNA at single extracellular vesicle level by droplet microfluidics for cancer diagnosis.

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.