A Single Excitation‐Duplexed Imaging Strategy for Profiling Cell Surface Protein‐Specific Glycoforms

Abstract This work develops a site‐specific duplexed luminescence resonance energy transfer system on cell surface for simultaneous imaging of two kinds of monosaccharides on a specific protein by single near‐infrared excitation. The single excitation‐duplexed imaging system utilizes aptamer modified upconversion luminescent nanoparticles as an energy donor to target the protein, and two fluorescent dye acceptors to tag two kinds of cell surface monosaccharides by a dual metabolic labeling technique. Upon excitation at 980 nm, only the dyes linked to protein‐specific glycans can be lit up by the donor by two parallel energy transfer processes, for in situ duplexed imaging of glycoforms on specific protein. Using MUC1 as the model, this strategy can visualize distinct glycoforms of MUC1 on various cell types and quantitatively track terminal monosaccharide pattern. This approach provides a versatile platform for profiling protein‐specific glycoforms, thus contributing to the study of the regulation mechanisms of protein functions by glycosylation.