J. Eur. Opt. Society-Rapid Publ. 22, 10( 2026) 99
Fig. 2.( a) Fluorescent detection of O‐GlcNAc via Tandem Glycan Labeling.( b) Fluorescent labeling technique for lipid and surfactant preformed vesicles.( c) Efficient labeling of vesicles with lipophilic fluorescent dyes via the Salt-Change method.
improved the selective recognition ability of probes by coupling fluorophores with biological targeting molecules such as antibodies [ 33 ], nucleic acid aptamers [ 34 ], and developing fluorescein derivatives [ 35, 36 ], which not only provides a powerful tool for studying biomolecular dynamics and photophysical properties, but also shows important application value in biomedical fields( such as anticancer drug research) [ 37 ].
A long-standing challenge in the O-GlcNAc field has been the lack of tools for specific, non-antibody-based detection. Traditional immunoassays, while widely used, often suffer from cross-reactivity and limited ability to distinguish O-GlcNAcylation from other post-translational modifications in complex samples. To address this specificity gap, the breakthrough work by Wu et al. [ 38 ] introduced a tandem glycan labeling strategy. This method converts the O-GlcNAc moiety into a fluorescently tagged epitope through sequential enzymatic steps, thereby enabling highly specific and sensitive detection independent of conventional antibodies, as shown in Figure 2a. This method achieves two-step labeling by galactosylating O-GlcNAc by B4GalT1 and then connecting the fluorescent moiety by ST6Gal1- mediated sialylation. The experimental validation showed that the method could efficiently detect O-GlcNAc modification in HEK293 nuclear extract and recombinant proteins( such as CK2, HIF1 a), and the signal was specific after pre-treatment with OGA / OGT. Compared with the traditional antibody method, this method avoids the side reactions of click chemistry, has a sensitivity of femtomolar level, and supports direct imaging by SDS-PAGE. This technology provides a new tool for the visualization and functional study of low abundance O-GlcNAc protein, reveals the potential O-GlcNAc regulatory mechanism of HIF1 a and other targets, and opens up a new way for glycobiology research.
In the field of extracellular vesicles( EVs), fluorescent labeling technology also plays an important role. Mousseau et al. [ 39 ] developed a universal fluorescent labeling technology based on the fat soluble carbocyanine dye
PKH67 for the tracking study of prefabricated liposomes and surfactant vesicles, as shown in Figure 2b. Thismethod avoids fluorescence quenching caused by dye aggregation by optimizing the molar ratio of dye to lipid, and does not require complex purification steps. The experimental verification showed that this technology could efficiently label neutral phospholipids( DPPC), negatively charged pulmonary surfactant( Curosurf) and positively charged double chain surfactant( TEQ) vesicles, and the size and surface potential of the vesicles remained stable after labeling. Through fluorescence microscopy and confocal imaging, the research team successfully tracked the interaction between nanoparticles and vesicles and their separation process in cells, visualized the adsorption behavior of cationic surfactants on cotton fibers, and revealed the characteristics of their deposition in the form of bilayers or vesicles. This technology provides a simple and efficient solution for vesicle tracking in biomedical and industrial applications.
However, conventional methods are often inefficient during labeling due to the low water solubility of the dyes, and the use of high concentrations of dyes is prone to problems such as nonspecific labeling and changes in vesicle size. To address these challenges, Cha et al. [ 40 ] proposed a vesicle fluorescence labeling technique based on salt concentration modulation, the Salt-Change Method, as shown in Figure 2c. The labeling efficiency of the lipophilic dye DiI was significantly enhanced by dynamically adjusting the NaCl concentration( low-salt condition promotes dye dispersion, and high-salt condition induces free dye aggregation) combined with filter membrane purification. Experimental validation showed that the method is applicable to cell-derived vesicles( CDVs), natural EVs, and synthetic liposomes, and that the size distribution and membrane protein function( such as antibody binding to CD63 / PD-1) of the labeled vesicles remain intact. In addition, themethodiscompatiblewithawiderangeofdyes( DiD, PKH67), is easy to perform, and reduces nonspecific labeling by lowering the dye concentration. This technique