Accurate and rapid quantification of PD-L1 positive exosomes by a triple-helix molecular probe

Programmed death ligand-1 (PD-L1) positive exosomes (P-Exo) have been widely used for tumor diagnosis. However, accurate and rapid quantification of P-Exo remains challenging due to the heterogeneity of clinical individuals and isolation techniques. In this study, the triple-helix molecular probe (THMP) coupled with high-affinity silica-based TiO₂ magnetic beads was used to isolate exosomes and to analyze the relative abundance of P-Exo in total exosomes (T-Exo). By employing this strategy, the entire analysis was completed within 70 min and the detection limit for P-Exo was 880 particles μL^-1. Additionally, the relative abundance of P-Exo in T-Exo (RA_P-Exo/T-Exo) was calculated from their fluorescence ratio, which could avoid errors due to differences in samples and separation methods, and identify 1.5 × 10³ P-Exo from 5 × 10⁶ T-Exo per microliter. RA_P-Exo/T-Exo values were not only effective in distinguishing healthy volunteers from breast cancer patients, but also highly positively correlated with the stage of breast carcinoma. Overall, this strategy opens a new avenue for rapid and quantitative analysis of P-Exo, providing an opportunity for precise diagnosis and prediction of treatment efficacy in cancer.