Ly Chi-Phong, Tran Nguyet Thuy, Toublanc Michaël, Delaunay Nathalie, Taverna Myriam, Mai Thanh Duc
Institut Galien Paris-Saclay, UMR CNRS 8612, Paris-Saclay University, 91400, Orsay, France.
Laboratoire des Sciences Analytiques, Bioanalytiques et Miniaturisation, UMR CBI 8231, ESPCI Paris, PSL University, 75005, Paris, France.
Talanta. 2025 Nov 1;294:128247. doi: 10.1016/j.talanta.2025.128247. Epub 2025 May 3.
We present in this study the development of digital capillary electrophoresis (DCE), together with a dual-stage on-line electrokinetic preconcentration protocol for hydrodynamic injection and capillary electrophoresis (CE) separation of N-glycans from a microdroplet. Several features that were not met in previous droplet-interfaced CE systems could now be realized with the DCE system that is based on a hybrid setup of micro-syringes and miniature pressure controllers. It allows i) working with a preprocessed sample volume as small as 500 nL, ii) and precise injection of a sub-microliter sample droplet into the capillary without penetration of oil inside nor current leakage during CE separation. Furthermore, the DCE was coupled with a new way of dual-stage electrokinetic preconcentration method combining large volume sample stacking with electroosmotic pump (LVSEP) and transient-isotachophoresis (tITP) to allow enrichment of the analytes from quasi-totality of the sample microdroplet to drastically boost the detection sensitivity. The DCE platform with the dual-stage LVSEP-tITP method brings a solution to overcome some major actual challenges in microscale electrophoresis, notably incompatibility of the working volumes and unsatisfactory detection sensitivity. To demonstrate the significance of DCE-LVSEP-tITP, the system and the dual-stage preconcentration protocol were applied for CE separation and fluorescent detection by LED induced fluorescence (LEDIF) of a labelled malto-oligosacharride ladder (MD Ladder) and N-glycans released from human IgG. With the best LVSEP-tITP conditions using the background electrolyte composed of triethanolamine (TEOA)/citric acid at pH 4.75 and ionic strength (IS) of 150 mM, the leading electrolyte composed of 8.0 μM APTS in deionized water and the terminating electrolyte composed of TEOA/citric acid at pH 3.0 and IS of 200 mM, excellent sample enrichment factors (SEFs) could be obtained for glucose oligomers with good repeatability on migration time and peak area (RSD <1.0 % and 5.0 %, respectively). Our approach offers sample enrichment factors (SEFs) up to 620 folds compared to that obtained with our CE-LIF approach for glycan analysis, and 2400 compared to that achieved with the reference capillary gel electrophoresis method with laser induced fluorescence detection (CGE-LIF), allowing to reach the detection and quantification limits for CE-LIF of glycans down to 0.03 and 0.1 ng/mL, respectively.
在本研究中，我们展示了数字毛细管电泳（DCE）的发展，以及一种双阶段在线电动预浓缩方案，用于从微滴中进行流体动力进样和毛细管电泳（CE）分离N - 聚糖。以前的液滴接口CE系统中未实现的几个功能，现在可以通过基于微注射器和微型压力控制器混合设置的DCE系统来实现。它允许：i）使用低至500 nL的预处理样品体积进行操作；ii）将亚微升样品液滴精确注入毛细管，在CE分离过程中油不会进入且无电流泄漏。此外，DCE与一种新的双阶段电动预浓缩方法相结合，该方法将大体积样品堆积与电渗泵（LVSEP）和瞬态等速电泳（tITP）相结合，以允许从几乎整个样品微滴中富集分析物，从而大幅提高检测灵敏度。具有双阶段LVSEP - tITP方法的DCE平台为克服微尺度电泳中的一些主要实际挑战提供了一种解决方案，特别是工作体积不兼容和检测灵敏度不理想的问题。为了证明DCE - LVSEP - tITP的重要性，该系统和双阶段预浓缩方案被应用于CE分离和通过LED诱导荧光（LEDIF）对标记的麦芽寡糖阶梯（MD阶梯）和从人IgG释放的N - 聚糖进行荧光检测。在最佳LVSEP - tITP条件下，使用pH 4.75、离子强度（IS）为150 mM的三乙醇胺（TEOA）/柠檬酸组成的背景电解质，去离子水中8.0 μM APTS组成的前导电解质，以及pH 3.0、IS为200 mM的TEOA/柠檬酸组成的终止电解质，可以获得葡萄糖寡聚物的优异样品富集因子（SEF），迁移时间和峰面积具有良好的重复性（RSD分别<1.0%和5.0%）。与我们用于聚糖分析的CE - LIF方法相比，我们的方法提供的样品富集因子（SEF）高达620倍，与参考毛细管凝胶电泳激光诱导荧光检测方法（CGE - LIF）相比高达2400倍，使得CE - LIF对聚糖的检测限和定量限分别低至0.03和0.1 ng/mL。
