Single-cell N-glycan analysis via laser-induced fluorescent capillary electrophoresis with femtomolar sensitivity

Glycans are involved in numerous biological processes such as cell-to-cell interactions, immune responses, and signaling pathways. Sensitive glycan analysis technology is essential for exploring cellular heterogeneity, analyzing tumor micro-sections, characterizing rare samples, and so on. However, analytical sensitivity is challenging because of the limited number of glycans in trace samples, lack of glycan amplification technology, or sample loss during the analysis workflow. This study employed capillary electrophoresis coupled with laser-induced fluorescence (CE-LIF) to analyze 8-aminopyrene-1,3,6-trisulfonic acid (APTS)-labelled N -glycans. Efforts were made to enhance analytical sensitivity by enhancing the detection sensitivity and limiting sample loss. A customized LIF system was proposed based on the specific spectrum of APTS-labeled glycans, achieving 3-orders sensitivity enhancement compared to commercial LIF system. In addition, an electrokinetic supercharging (EKS) protocol was developed for sample preconcentration, providing another 3-orders sensitivity enhancement compared to hydrodynamic assisted sample loading. By applying the LIF and the EKS protocol to CE-LIF, the limit of detection ( LOD ) was found to be 4.7 femtomolar for standard glycan samples. N -glycans from HeLa cells were analyzed and annotated with glucose unit values with a relative standard deviation of less than 1.4 %. Single-cell N -glycan analysis was successfully performed using single and double HeLa cells. This CE-LIF method showed high sensitivity for APTS-labeled N-glycans , indicating great potential for trace sample analysis. We believe it will have a wide application in biological studies, pharmaceutical development and clinical diagnosis. • Femtomolar level limit of detection and single-cell analysis was achieved in CE-LIF. • A customized LIF system was developed with 3-order sensitivity enhancement. • A sample preconcentration method was developed and enhanced the sensitivity by another 3-order.