编程细胞凋亡配体-1（PD-L1）阳性外泌体（P-Exo）已被广泛用于肿瘤诊断。然而，由于临床个体和分离技术的异质性，对P-Exo的准确和快速定量分析仍面临挑战。在本研究中，利用三重螺旋分子探针（THMP）结合高亲和力硅基TiO2磁珠来分离外泌体并分析P-Exo在总外泌体（T-Exo）中的相对丰度。通过采用这种策略，整个分析过程在70分钟内完成，P-Exo的检测限为880颗μL-1。此外，通过他们的荧光比计算P-Exo在T-Exo中的相对丰度（RAP-Exo/T-Exo），可以避免由于样品和分离方法的差异而产生的误差，并且每微升可以鉴定出5×106 T-Exo中的1.5×103 P-Exo。RAP-Exo/T-Exo值不仅能有效区分健康志愿者和乳腺癌患者，而且与乳腺癌的分期高度正相关。总的来说，这种策略为快速定量分析P-Exo开辟了一条新的途径，为精确诊断和治疗效果预测提供了机会。版权所有©2023 Elsevier B.V.，保留所有权利。

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.Copyright © 2023 Elsevier B.V. All rights reserved.