ELECTROKINETIC SEPARATIONS ON MICROFLUIDIC DEVICES FOR N-GLYCAN PROFILING

Abstract

Microfluidic devices have been employed for various chromatographic and electrophoretic separations of biomolecules. However, the inherent complexity of analyzing real biological samples requires faster and more efficient separation techniques. N-glycans are essential molecules in many living systems and may become important biomarkers for cancer and other diseases in humans. In this work, we developed several microfluidic platforms for separation and analysis of N-glycans. On microfluidic devices, channels with high separation efficiencies can be incorporated while maintaining a small footprint. However, incorporating turns with small radii of curvature can cause significant band broadening. We explored two options to minimize the turn-induced band broadening: a spiral channel with a large radius of curvature and serpentine channels with asymetrically tapered turns. The latter serpentine design offers a more compact footprint than the spiral design. Using separation lengths of ~20 cm and electric field strengths up to 1500 V/cm, analysis times were less than 1.2 min, and separation efficiencies were between 500,000 and 655,000 plates for the N-glycans. These high efficiencies are necessary to separate structural isomers that exist in these samples. A direct comparison between microchip and capillary electrophoresis demonstrated the separation performance on microchips was as good as or better than on capillaries. Statistical analysis of the glycan profiles derived from blood serum samples revealed differences between healthy individuals and cancer patients, and relevant glycan structures may be used as biomarkers for early stage screening and prognosis. To further increase the separation peak capacity, microfluidic platforms with serial-to-parallel interfaces were evaluated for two-dimensional N-glycan profiling. Three designs and operation modes were investigated, and an interface with a gated valve provided the highest performance and reproducibility. Capillary electrochromatography with monolithic stationary phases and capillary electrophoresis were coupled together as first and second dimensions, respectively.