acDEP-assisted microfluidic chip for ultrasensitive detection of urinary exosomes for non-invasive hepatocellular carcinoma diagnosis.

Early detection of hepatocellular carcinoma (HCC) remains challenging due to the lack of sensitive and non-invasive diagnostic strategies. Urinary exosomes (or small extracellular vesicles, sEVs) represent a promising biomarker source, but technical challenges remain in achieving efficient and rapid separation coupled with immediate high-sensitivity analysis. To address this, we developed an alternating current dielectrophoresis-assisted microfluidic platform integrated with amphiphilic dendrimer supramolecular probes (ADSP) for rapid and sensitive detection. In this system, a non-uniform alternating electric field generated by comb-shaped electrodes actively drives exosomes toward an ADSP-modified nitrocellulose (NC) membrane via positive dielectrophoresis, while amphiphilic amphotericin B (AMB)-mediated supramolecular interactions enable stable capture through insertion into the exosomal lipid bilayer. The combined effect of electric-field enrichment and probe-mediated recognition significantly improves capture efficiency with a limit of detection (LOD) of 6 exosomes/μL, requiring only 52 μL of sample and 9 min for injection within a simple chip structure. Key operational parameters, including voltage, frequency, flow rate, and sample volume, were systematically optimized to maximize enrichment performance. The platform enabled highly sensitive fluorescence signal detection of exosomal biomarkers and successfully distinguished urine samples from HCC patients and healthy controls. Benefiting from the synergistic integration of electric-field-driven enrichment and supramolecular probe recognition, this strategy provides a simple, valid, and rapid approach for urinary exosome analysis, indicating its applicability to non-invasive liquid biopsy-based cancer detection.