In-Capillary Integrated CE-LIF Platform with Bioinformatics-Guided Exosome Capture and Isothermal Amplification for Ultrasensitive Multiplexed Detection of HCC-Derived Exosomal MicroRNAs

Exosomal microRNAs (Exo-miRNAs) are promising liquid-biopsy biomarkers for noninvasive cancer detection; yet due to their short length, low abundance, and high sequence homology, the determination of Exo-miRNAs suffers from insufficient sensitivity, limited multiplexing, and operational complexity, impeding their high-throughput clinical applications. Here, we introduce an in-capillary integrated inline analytical platform specifically tailored for hepatocellular carcinoma (HCC)-derived Exo-miRNAs. The platform combines four functions within a single capillary: (i) tumor-specific exosome capture via an α-fetoprotein (AFP) aptamer, (ii) isothermal signal amplification using catalytic hairpin assembly coupled with exonuclease III (CHA-ExoIII), (iii) capillary electrophoresis (CE) separation, and (iv) laser-induced fluorescence (LIF) detection. Through bioinformatic analysis, AFP is identified as a selective membrane marker, enabling direct in-column enrichment of HCC exosomes from complex samples without labor-intensive isolation. Differential expression of miR-21, miR-223, and miR-122 in HCC cells and their exosomes validated a combined diagnostic panel. CHA-ExoIII amplification achieved femtomolar sensitivity, while CE-LIF provided rapid, high-resolution quantification of multiple targets in a single run. The results confirm the platform’s suitability for distinguishing HCC-derived exosomes from normal counterparts in complex biological matrices. The streamlined in-capillary workflow presented in the study, i.e., exosome capture, nucleic-acid amplification, CE separation and LIF detection, combined with a commercial kit-based exosome extraction step prior to analysis, would pave the way to sensitive, selective and rapid solutions for early tumor screening and liquid-biopsy analysis.