Upconversion Luminescent Nanoprobes in Conjunction with Rolling Circle Amplification for Phenotypic Profiling of Exosome from Pancreatic Cancer

Exosomes have emerged as potential biomarkers for non-invasive diagnosis and real-time monitoring of disease due to their ability to carry bio-molecular information from their source cells and stability in various body fluids. However, existing detection methods face challenges of insufficient specificity, limited sensitivity, and difficulty in distinguishing exosomes from different cellular origins. To address this, this study developed a fluorescent biosensing platform based on silica-coated upconversion nanoparticles (UCNPs@SiO2) for characterization of exosomes derived from pancreatic cancer cells that simultaneously overexpress epidermal growth factor receptor (EGFR) and epithelial cell adhesion molecule (EpCAM). The platform employed a cascade signal amplification strategy. Firstly, exosomes were captured using magnetic beads conjugated with anti-CD63 antibodies. Subsequently, specific recognition of EGFR and EpCAM was achieved via DNA aptamers, triggering rolling circle amplification (RCA) to generate long-chain DNA products containing repetitive sequences. Finally, UCNPs-labeled DNA probes complementary to RCA products were added. After incubation and magnetic separation, the fluorescent signal of free UCNPs in the supernatant was detected. Experimental results showed that the supernatant fluorescence intensity decreased with increasing exosome concentration, exhibiting a negative correlation response. Additionally, semi-quantitative naked-eye analysis could be performed by observing the degree of magnetic bead aggregation, with the lowest visually distinguishable concentration for EGFR- and EpCAM-positive exosomes being 3.0×103 particles/mL and 6.0×103 particles/mL, respectively. The analytical method established in this study enabled high-sensitivity, dual-protein phenotyping of exosomes from specific cellular origins, providing a novel methodological support for non-invasive diagnosis and therapeutic monitoring of pancreatic cancer.