Faculty of Chemistry, Northeast Normal University, Changchun 130024, Jilin, China.
Department of Chemistry and the MOE Key Laboratory of Spectrochemical Analysis and Instrumentation, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China.
J Chromatogr A. 2014 Jul 25;1352:80-6. doi: 10.1016/j.chroma.2014.05.058. Epub 2014 May 27.
A novel method using particle-packing technique to fabricate capillary electrophoresis (CE)-based immobilized enzyme reactor (IMER) was accomplished by utilizing perfusive silica single particles as the frits and large-pore beads as the enzyme supports. The fabrication procedure is rapid and simple; the length and enzyme loading amount of the CE-IMERs could be easily adjusted. Performance and feasibility of the CE-IMERs were investigated using on-line trypsin digestion as the model enzyme reaction. High reproducible on-line enzyme assay was demonstrated with RSD less than 4.1% and 3.8% for peak area and migration time of the substrate and product over 100 consecutive runs, respectively. The enzyme can still maintain the activity for at least 10 days, indicating remarkably stability of the CE-IMERs. The CE-IMERs were successfully applied for accurate analysis of trypsin inhibition as well as on-line digestion of standard proteins (myoglobin and BSA). The present method provides a new interesting alternative to open-tubular and monolithic CE-IMERs, thus expands the application of the CE technique for on-line enzyme assay and analysis and characterization of peptides and proteins.
一种使用颗粒填充技术制备毛细管电泳（CE）固定化酶反应器（IMER）的新方法，是利用可渗透硅单颗粒作为过滤片和大孔珠作为酶载体来实现的。该制备过程快速且简单；CE-IMER 的长度和酶负载量可以轻松调节。通过在线胰蛋白酶消化作为模型酶反应来研究 CE-IMER 的性能和可行性。结果表明，在 100 次连续运行中，底物和产物的峰面积和迁移时间的重现性好，相对标准偏差（RSD）均小于 4.1%和 3.8%，可以进行高重复性的在线酶分析。此外，该酶至少可以保持 10 天的活性，表明 CE-IMER 具有显著的稳定性。该 CE-IMER 还成功地应用于准确分析胰蛋白酶抑制以及标准蛋白质（肌红蛋白和 BSA）的在线消化。本方法为开管和整体 CE-IMER 提供了一种新的有趣选择，从而扩展了 CE 技术在在线酶分析以及肽和蛋白质的分析和特性方面的应用。
