Flexible Dual-Apt Scaffolds Reveal gPD-L1-sEVs in Breast Cancer

The PD-1/PD-L1 blockade is a cornerstone of breast cancer immunotherapy, yet predictive biomarkers for immunotherapy outcomes remain scarce, restricting its clinical benefit. Glycosylated PD-L1 (gPD-L1) regulates tumor progression, immune suppression, and immune-checkpoint blockade (ICB) response, but it remains undetectable by conventional immunohistochemistry (IHC) due to glycan-mediated epitope shielding. This "detection blind spot" largely undermines current ICB biomarker prediction. To address this unmet clinical need, we developed an ultrasensitive CAP-g bioplatform with a novel CF/DPEDOT:PSS/Au nanoflexible scaffold. It integrates a dual-recognition-driven proximity cascade amplification for accurate gPD-L1 on small extracellular vesicle (gPD-L1-sEV) detection. We constructed this three-dimensional (3D) CF/DPEDOT:PSS/Au nanonetwork scaffold electrode, where π-π stacking synergistically minimizes charge-transfer resistance to improve electron transfer efficiency and maximizes the effective reaction area. Co-anchoring of a PD-L1 aptamer and a glycan probe further triggers hybridization chain reaction (HCR) for signal amplification, and this integrated design outperforms conventional electrochemical or immunological detection strategies. The CAP-g platform achieves an ultralow limit of detection (LOD) of 52 particles/mL, which is 1-2 orders of magnitude lower than existing gPD-L1-sEV or PD-L1-sEV detection methods, and a broad linear dynamic range of 10³-10⁸ particles/mL. Clinical validation in breast cancer patients and healthy controls showed robust discrimination between the two groups (P < 0.0001), with high specificity and reproducibility. This work overcomes the technical bottleneck of glycosylation interference in gPD-L1 detection, establishes a new liquid-biopsy paradigm for gPD-L1-sEV detection, and provides a reliable tool for predicting ICB efficacy in breast cancer, thus laying a critical foundation for personalized immunotherapy.