Digital capillary electrophoresis with dual preconcentration for sub-microliter samples: A proof-of-concept for glycan analysis.

Authors

Chi-Phong Ly, Nguyet Thuy Tran, Michaël Toublanc, Nathalie Delaunay, Myriam Taverna, Thanh Duc Mai

Published in

Talanta. Volume 294. Pages 128247. May 03, 2025. Epub May 03, 2025.

Abstract

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.

PMID:
40334513
Bibliographic data and abstract were imported from PubMed on 08 May 2025.

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