Single-tube total analysis system for ratiometric detection of exosomal miRNAs in breast cancer diagnosis

Although exosomal microRNAs (Exo-miRs) show great promise as stable and disease-specific biomarkers for early cancer detection, current detection workflows remain technically fragmented into multiple discrete steps (exosome isolation, miRNA amplification, and signal detection), requiring inter-step sample transfers. In addition, most existing platforms lack effective normalization strategies, hindering accurate cancer diagnosis because of intersample variation in exosome yield. Here, we developed a single-tube Exo-miRs total analysis system (ST-Exo-TAS) that integrates the entire Exo-miR workflow within a single reaction tube, enabling normalized analysis of target Exo-miRs. The system employs promoter-enriched gold nanoparticles conjugated with anti-CD63 antibodies (Pr/CD63Ab/AuNPs), which serve a dual function: isolating exosomes via immunoaffinity under low-speed centrifugation (6000 g, 10 min) and acting as promoter scaffolds for one-pot isothermal elongation and amplification (OPIEA). Following isolation, the captured Exo-miRs are directly subjected to OPIEA at 37 °C using SplintR ligase and T7 polymerase, generating light-up aptamers templated by miR-21 (target) and miR-U6 (internal control). This enables ratiometric dual-fluorescence detection via simple dye addition. ST-Exo-TAS demonstrated high sensitivity, with limits of detection of 1.179 fM for miR-21 and 843.9 aM for miR-U6, and high specificity within a short assay time (sample-to-answer = 150 min). Furthermore, the system was validated using preclinical and clinical samples. Breast cancer cases were successfully distinguished by quantifying Exo-miR-21 expression normalized to that of miR-U6, which helped minimize intersample variation in exosome yield (AUC = 1). This rapid, compact, and user-friendly platform offers a powerful tool for early breast cancer diagnosis and holds strong potential in point-of-care diagnosis technology.