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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 (RAP-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. RAP-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.
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•A Triple-helix molecular probe is proposed for quantifying PD-L1 positive exosome (P-Exo).•The silicon-based TiO2 magnetic beads is used for rapid and efficient capture of exosomes.•The proposed relative abundance of P-Exo in T-Exo enables precise differentiation of tumor progression.•This method could identify 1.5 × 103 P-Exo from 5 × 106 total exosomes (T-Exo) per microliter.