Lectin-mediated in situ rolling circle amplification on exosomes for probing cancer-related glycan pattern

Exosomal surface glycans play important roles in microvesicle protein sorting and exosome-cell interactions, and also provide promising biomarkers for various diseases. However, in situ detection techniques for exosomal glycans are largely lacking. In this work, an exosomal array is fabricated for probing cancer-related exosomal glycan signatures by lectin recognition-mediated in situ rolling circle assembly of fluorophore-labeled DNA. Different from commonly used lectin array, the proposed strategy enables the direct and amplified conversion of glycan recognition signals to fluorescence detection signals. Focusing on tumor-associated glycans including sialic acids, fucose and truncated O-glycans, the method has been used not only to compare glycan patterns between exosomes with different origins, but also to reveal the specific exosomal glycan characteristics compared to their parent cells. The limits of detection were identified to be 5.4 × 10⁶ and 1.3 × 10⁶ particles mL^-1 for HeLa and PANC-1 exosomes, respectively. The dynamic ranges were 4.7 × 10⁵ to 4.7 × 10⁸, 4.7 × 10⁸ to 4.7 × 10⁹ for HeLa exosomes, and 4.7 × 10⁵ to 1.2 × 10⁹, 1.2 × 10⁹ to 4.7 × 10⁹ particles mL^-1 for PANC-1 exosomes. The remodeling of exosomal glycans can also be monitored as demonstrated on the cleavage of sialic acids under sialidase treatment. It could be anticipated that this strategy would become a powerful tool for development of exosome-based glyco-biomarkers and elucidation of biological significance of exosomal glycans.