芯片自由流梯度电场电泳及其应用

Micro free flow gradient electrical field electrophoresis and its application

尽管在垂直的电场和流体场作用下,采用芯片自由流电泳(μ-FFE)可实现样品的连续微分离和制备,但是由于在运行过程中,存在分析物的区带展宽问题,会直接影响样品的分离效果.在本文中,在施加固定电压的情况下,通过向和分离缓冲液相同的电极缓冲液中添加硫酸钠的方法,在分离腔内形成了梯度电场.通过对罗丹明B和甲基绿混合物的分离发现,在均一电场下,施加400V分离电压,混合物需2min才能完全分离;甲基绿的区带宽度为3.8mm,与罗丹明B的分辨率是3.2.在向电极缓冲液中添加5mmol/L硫酸钠形成的电场梯度下,施加300V的分离电压,两种染料可在10s内完成分离;在20s时,甲基绿的区带宽度被压缩到0.5mm,检测灵敏度提高了7倍以上;与罗丹明B的分辨率可达到16.2.此外,该方法还被用于牛血清白蛋白的富集.与施加均一电场相比,蛋白质的检测灵敏度得到了显著提高.上述结果表明,通过在μ-FFE中引入梯度电场,可有效提高样品的分辨率、检测灵敏度和分析速度.

Although samples could be continuously separated and prepared by μ-FFE in microscale with orthogonal electrical field and hydrodynamic field, band broadening of analytes is still a serious problem that might affect resolution. In this work, with a fixed applied voltage, a gradient electrical field （GEF） was formed in the separation chamber by adding 5 mmol.L^-1 sodium sulfate into electrode buffer, although other components were the same as those in separation buffer. Without GEF added, a mixture of methyl green and Rhodamine B was separated completely under 400 V applied voltage for 2 min. The band width of methyl green was 3.8 mm, and the resolution of methyl green and Rhodamine B was only 3.2. With GEF applied, the baseline separation of such two dyes was obtained with 300 V applied voltage for 10 s. In addition, the band width of methyl green was concentrated to 0.5 mm in 20 s, and the detection sensitivity was improved by over 7 times. Furthermore, by such a method, the detection sensitivity of BSA was increased evidently. All these results demonstrated that with the resolution, the separation speed and the detection sensitivity of samples could be improved significantly by μ-FFE with GEF applied.