Tracing Tumor‐Derived Exosomal PD‐L1 by Dual‐Aptamer Activated Proximity‐Induced Droplet Digital PCR

Tumor‐derived exosomal proteins have emerged as promising biomarkers for cancer diagnosis, but the quantitation accuracy is hindered by large numbers of normal cell‐derived exosomes. Herein, we developed a dual‐target‐specific aptamer recognition activated in situ connection system on exosome membrane combined with droplet digital PCR (ddPCR) (TRACER) for quantitation of tumor‐derived exosomal PD‐L1 (Exo‐PD‐L1). Leveraging the high binding affinity of aptamers, excellent selectivity of dual‐aptamer recognition, and the high sensitivity of ddPCR, this method exhibits significant sensitivity and selectivity for tracing tumor‐derived Exo‐PD‐L1 in a wash‐free manner. Due to the excellent sensitivity, the level of tumor‐derived Exo‐PD‐L1 detected by TRACER can distinguish cancer patients from healthy donors, and for the first time was identified as a more reliable tumor diagnostic marker than total Exo‐PD‐L1. The TRACER strategy holds great potential for converting exosomes into reliable clinical indicators and exploring the biological functions of exosomes. We developed a dual‐target‐specific aptamer recognition system combined with droplet digital PCR for precise quantitative analysis of exosomal PD‐L1 (Exo‐PD‐L1). This method can distinguish tumor‐derived from non‐tumor‐derived Exo‐PD‐L1, holding great potential for the analysis of exosome subtypes and offering unprecedented opportunities for the study of the biological functions of exosomes and their conversion into reliable clinical indicators.