Ultrasensitive Multiplex Detection of Lung Cancer EV-Associated Immune Checkpoints Using a Mesoporous Gold Enhanced SERS Biosensor

Abstract

Immune checkpoint proteins (ICPs) play a critical role in tumor immune evasion and have emerged as key biomarkers for cancer diagnosis, treatment selection, and monitoring immunotherapy responses, particularly in lung cancer. Frequent assessment of ICP expression is essential for guiding therapeutic decisions and evaluating treatment efficacy. Extracellular vesicles (EVs), secreted into the bloodstream by cancer cells, mirror the molecular profile of their cells of origin, including surface-expressed ICPs, offering a minimally invasive window into tumor biology. However, conventional assays lack the sensitivity and specificity to detect ICPs on heterogeneous EV populations. Here, we present a highly sensitive and integrated biosensing platform based on surface-enhanced Raman scattering (SERS) coupled with a nanostructured mesoporous gold chip for multiplexed detection of ICPs on lung cancer-derived EVs. The platform incorporates an anti-EGFR-functionalized mesoporous gold surface to selectively capture tumor-derived EVs and utilizes SERS nanotags for the simultaneous detection of four key ICPs: PD-L1, B7H4, CD276, and CD80. The highly ordered and densely packed pores of the 3D mesoporous gold nanostructure increase the surface area for antibody immobilization, while simultaneously amplifying SERS signal intensity through localized plasmonic hotspots - together enabling ultra-sensitive and highly specific detection of EV-associated biomarkers. Using EVs derived from the HCC827 lung cancer cell line, our platform successfully identified target EVs within a complex background and profiled ICP expression patterns. Notably, CD276, PD-L1, and B7H4 exhibited dominant expression on EV surfaces, with marked alterations in their levels following EGFR inhibitor treatment—highlighting the dynamic nature of immune modulation in response to therapy. These findings underscore the clinical utility of the mesoporous gold-integrated SERS platform for non-invasive lung cancer diagnostics and longitudinal monitoring of immunotherapy-induced immune modulation.