Holland Lisa A, Gattu Srikanth, Crihfield Cassandra L, Bwanali Lloyd
C. Eugene Bennett Department of Chemistry, West Virginia University, Morgantown, WV 26506, United States.
C. Eugene Bennett Department of Chemistry, West Virginia University, Morgantown, WV 26506, United States.
J Chromatogr A. 2017 Nov 10;1523:90-96. doi: 10.1016/j.chroma.2017.06.038. Epub 2017 Jun 16.
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.
一种热响应性纳米凝胶用于在分离毛细管中创建酶和凝集素的固定区域。一旦在毛细管中形成图案，分析物就会被驱动通过该区域，如果使用酶，分析物会在该区域转化为特定产物，如果使用凝集素，分析物会被捕获。分析完成后，这些固定区域很容易被排出，然后在毛细管中重新形成图案。纳米凝胶与酶和凝集素兼容，并提高了半乳糖苷酶的稳定性，从而能够更经济高效地使用可深入了解聚糖结构的生物试剂。使用固定区域的一个特点是，反应时间可以通过区域长度、控制分析物迁移率的外加电场或在分析物位于该区域时通过切换外加电压的极性进行电泳混合来控制。对提高酶性能的温度、外加电压和固定区域长度等因素进行了表征。在毛细管电泳中联合使用酶和凝集素是一种新策略，可利用纳升体积的酶和凝集素来推进聚糖的快速自动化分析。通过鉴定N-聚糖中β(1-3)-连接的半乳糖，证明了在毛细管电泳中使用酶和凝集素固定区域的适用性。
