Abstract
Liquid biopsy is a transformative, noninvasive tool for cancer diagnosis and monitoring, with the potential to revolutionize personalized medicine. In this study, we introduce an advanced liquid biopsy platform for highly sensitive and selective detection of extracellular vesicle (EV) microRNAs (miRNA-21 and miRNA-23a) as biomarkers for colorectal cancer. The platform combines two innovations: (1) gold nanoarrays with epithelial cell adhesion molecule (EpCAM)-specific aptamers to selectively isolate tumor-derived EVs from plasma and (2) CRISPR/Cas13a-encapsulated liposomes that fuse with EVs to form nanoscale reactors. Upon fusion, the CRISPR/Cas13a complex is activated by target miRNA, triggering trans-cleavage of RNA reporters and generating an amplified fluorescence signal for enhanced detection sensitivity. The assay achieves a linear detection range of 10 to 106 EVs/μL and a detection limit of 2.5 × 101 EVs/μL on the gold nanoarray. Its performance relies on three strategies: (i) EpCAM-mediated tumor EV enrichment, (ii) CRISPR/Cas13a-based collateral activity for ultrasensitive miRNA detection, and (iii) fluorescence signal enhancement via localized nanoreactors. Validation with a 2D SW480 cell model, a 3D vascularized tumor spheroid (VTS) model, and clinical plasma samples confirms diagnostic accuracy, with miRNA quantification comparable to RT-qPCR but without the need for labor-intensive RNA extraction and amplification. By integrating nanotechnology with CRISPR-based diagnostics, this platform bridges research and clinical translation, improving diagnostic precision and streamlining workflows. Future development will focus on multiplexed biomarker detection and single-EV analysis to reveal insights into EV heterogeneity and function in cancer. This technology supports the application in precision oncology, offering a tool for early detection, treatment monitoring, and therapeutic decision-making.