Lectin Recognition-Based In Situ Detection Of Tumor-Related Glycans On Cell Surface And Exosomes

Glycosylation is one of the most common post-translation modifications of proteins for most eukaryotic cells.It plays an important regulatory role for multiple life processes.The expression of glycans on cell surface is involved in cell metabolism,apoptosis and division,and is closely related to the interactions between cells,and also the progress of diseases.Thus the study of cell glycosylation has attracted more and more interest.Since glycans on protein peptide backbone play an important role in guiding proteins to target cell compartments,the glycosylation may exert potential influence on the protein sorting to membrane microdomains and thus the cargo constitution of exosomes.Thus the glycan signature of exosomes provides a valuable handle for understanding of the complicated mechanism of microvesicle protein sorting,and also the recognition and targeting tendency between exosomes and recipient cells.Considering the close relationship between exosomes with exosome-forming cells,the exosome surface glycan expression may reflect the glycome of the parent cells,suggesting the potential role of exosomes as carriers of glyco-biomarker.In this work,the glycan expression on cells and exosomes were analyzed using lectins that display specific binding toward glycans as recognition motifs.A sensitive,multichannel method for detection of glycan expression on exosomes was constructed by in situ rolling circle assembly of probes on exosomal array.Besides,using lipid-functional UCNP as a fluorescent donor that can be inserted into the cell membrane,a strategy for simultaneous imaging of two kinds of glycans on cell membranes based on dual-channel luminescent resonance energy transfer(LRET)was developed.1.Lectin-Mediated In Situ Rolling Circle Amplification on Exosomes for Probing Cancer-Related Glycan PatternIn this work,an exosomal array is fabricated for sensitive and multiplexed probing cancer-related exosomal glycan signatures by lectin recognition-mediated in situ rolling circle assembly of fluorophore-labeled DNA on exosomal glycans.Different from the existing methods,the proposed strategy enables the direct and amplified conversion of glycan recognition signals to fluorescence detection signals without pre-labeling or lysis of exosomes.The signal amplification module has been introduced into the detection of exosomal glycans for the first time in our work.Focusing on tumorassociated 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 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.2.UCNP-Based Duplexed-LRET Strategy for Simultaneous Detection of Dual Glycans on Cell SurfaceA single excitation-dual emission fluorescence "beacon" was fabricated on cell surface by inserting lipid-functionalized UCNP into cell membranes,which can be excited by near-infrared light(980 nm).After labeling different glycans on the cell surface by lectins modified with different fluorescence acceptors,the LRET could occur between the UCNP beacon and different dyes within a certain distance.Thus a duplexed channel imaging method was developed for cell surface glycans.Sialic acids and T antigens,which are closely related to tumors,were selected as the target glycans.And the problem of acceptor bleeding through in the common FRET processes was avoided through the up-conversion process.Using the "common fluorescence donor" design,simultaneous detection and relative quantification of two glycans on the cell membrane could be achieved.This method provides a universal platform for simultaneous detection of multiple biomolecules on cell surfaces.