Capillary electrophoresis with stationary nanogel zones of galactosidase and Erythrina cristagalli lectin for the determination of β(1-3)-linked galactose in glycans

A thermally responsive nanogel is used to create stationary zones of enzyme and lectin in a separation capillary. Once patterned in the capillary, analyte is driven through the zone, where it is converted to a specific product if an enzyme is used or captured if a lectin is used. These stationary zones are easily expelled after the analysis and then re-patterned in the capillary. The nanogel is compatible with enzymes and lectins and improves the stability of galactosidase, enabling more cost-effective use of biological reagents that provide insight into glycan structure. A feature of using stationary zones is that the reaction time can be controlled by the length of the zone, the applied field controlling the analyte mobility, or the use of electrophoretic mixing by switching the polarity of the applied voltage while the analyte is located in the zone. The temperature, applied voltage, and length of the stationary zone, which are factors that enhance the performance of the enzyme, are characterized. The combined use of enzymes and lectins in capillary electrophoresis is a new strategy to advance rapid and automated analyses of glycans using nanoliter volumes of enzymes and lectins. The applicability of this use of stationary zones of enzyme and lectin in capillary electrophoresis is demonstrated with the identification of β(1-3)-linked galactose in N-glycan.