Immunofluorescence Analysis for Simultaneous Liquid Biopsy of Multiple Hepatocellular Carcinoma Markers Using a...

Hepatocellular carcinoma (HCC) presents a global health burden due to its asymptomatic early course and aggressive progression. Conventional ultrasound often detects HCC at intermediate or advanced stages, beyond optimal intervention windows. This diagnostic limitation highlights the critical need for highly sensitive early detection methods. Although microfluidic platforms offer advantages in throughput and integration, their current focus on single biomarkers restricts diagnostic accuracy and clinical applicability. This study introduces the "Pro-Exo" chip, an innovative liquid-biopsy platform where antibody-conjugated microbeads disrupt laminar flow to improve binding efficiency, while multichannel parallel detection enables simultaneous analysis of multiple HCC biomarkers. This platform enables concurrent analysis of plasma proteins (DCP, GP73, AFP) and exosomal surface markers (CD9+CD81, EpCAM, GPC3) from minimal plasma volumes. By integrating different biomarker categories, it overcomes the limitations of single-analyte detection technologies. Validation studies demonstrated that the assay requires only 50 μL plasma per chamber and can be completed within 60 min, with low detection limits for all target biomarkers. Receiver operating characteristic analysis confirmed that single-marker detection provides insufficient diagnostic power, whereas combined plasma protein and exosomal marker detection significantly improved the accuracy of HCC identification. The Pro-Exo Chip establishes a robust platform for rapid, sensitive multibiomarker liquid biopsy, effectively addressing critical needs in early HCC detection. This integrated methodology not only advances cancer diagnostic strategies but also highlights the substantial clinical value of exosomal biomarkers, offering significant implications for both translational research and clinical practice.