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Life sciences · Journal article
Analytical Chemistry · November 2, 2026
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Abstract Multiplexed vibrational imaging holds great potential for tissue molecular analysis, yet fully standardized workflows suitable for routine clinical histopathology remain scarce. Herein, we report a clinically applicable methodology using monodisperse triple-bond Raman polymer nanoparticles (∼50 nm). This approach integrates one-step labeling and single-scan acquisition to achieve high-performance multiplexed Raman imaging of 5 protein markers in hepatocellular carcinoma (HCC) tissues. Targeting hepatitis B virus (HBV)-associated HCC, our biomarker panel combines the viral protein HBx with 4 clinically used indicators: AFP, GS, GPC3, and CD34. We further reveal a clinically critical dissociation: several high-grade HBV-HCC specimens exhibit markedly elevated oncogenic markers with comparatively low HBx expression, consistent with antiviral therapy suppressing viral protein production without reversing tumor aggressiveness. Taking advantage of the ultranarrow, well-resolved signals generated by triple-bond tags within the biological silent region (1800−2800 cm−1), we achieve high-fidelity spatial mapping and quantitative analysis. Unlike conventional pathological methods relying on morphological observation, clinical manifestations, or single-marker detection, this technique simultaneously evaluates viral etiology, tumor progression, and metastatic potential, effectively reducing misclassification risk in patients with discordant viral and oncogenic profiles, and completes the entire assay within approximately 24 h. This study establishes a rapid and reliable histopathological platform that integrates pathogen identification and comprehensive cancer characterization, demonstrating substantial value for both cancer research and clinical diagnostics.