Accurate Phenotyping at the Single-Exosome Level Using a Proximity-Ligation RCA-Based Light-Scattering Counting Platform for Breast Cancer Diagnosis

Tumor-derived exosomes (TEXs), carrying rich proteomic information, have shown great promise as noninvasive biomarkers for cancer detection. However, their inherent heterogeneity, relatively low abundance, and significant interference from non-TEXs in biological fluids pose considerable challenges for accurate and sensitive TEX quantification. To address this, we developed a highly sensitive and specific light-scattering counting platform based on proximity-ligation-mediated rolling circle amplification (RCA), enabling single-particle detection of exosomes. The approach employs dual aptamers that bind specifically to a single exosome, triggering proximity ligation and RCA. This process promotes the aggregation of small gold nanoparticles (AuNPs), generating a strong turn-on light-scattering signal from an initial near-zero background, which is then visualized as diffraction-limited spots. By using a single microbead as the signal enrichment platform to concentrate all exosome-derived signals, we achieved a detection sensitivity as low as 1 particle/μL. Furthermore, conjoint analysis of pairwise combinations of multiple exosomal proteins significantly improved diagnostic specificity, allowing precise discrimination between breast cancer patients and healthy individuals using just 1 μL of clinical plasma. By targeting other cancer-specific biomarkers, this platform can be readily adapted to detect other cancers, offering a versatile and scalable tool for early cancer diagnosis in the field of liquid biopsy.