Droplet Microfluidics with Capillary Electrophoresis for Glycan Biomarker Analysis.

Several glycoproteins are validated biomarkers of various diseases such as cancer, cardiovascular diseases, chronic alcohol abuse, or congenital disorders of glycosylation (CDG). In particular, CDG represent a group of more than 150 inherited diseases with varied symptoms affecting multiple organs. The distribution of glycans from target glycoprotein(s) can be used to extract information to help the diagnosis and possibly differentiate subtypes of CDG. Indeed, depending on the glycans and the proteins to which they are attached, glycans can play a very broad range of roles in both physical and biological properties of glycoproteins. For glycans in general, capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) has become a staple. Analysis of glycans with CE-LIF requires several sample preparation steps, including release of glycans from the target glycoprotein, fluorescent labeling of glycans, and purification of labeled glycans. Here, we describe the protocol for glycan sample treatment in a microfluidic droplet system prior to CE-LIF of labeled glycans. The microfluidic droplet approach offers full automation, sample, and reagent volume reduction and elimination of contamination from external environment.

Lab-in-droplet: From glycan sample treatment toward diagnostic screening of congenital disorders of glycosylation.

We present in this study a new microfluidic droplet platform, named Lab-in-Droplet, for multistep glycoprotein sample treatment. Several operations are required for the sample treatment of a given glycoprotein to profile its N-glycans. In our case, all preparation steps for the analysis of N-glycans from glycoproteins could be realized in an automatic manner and without cross contamination. This could be achieved through several features that are not met in previous droplet setups, notably full automation, droplet sensing and heating. The magnetic tweezer technology was employed to manipulate (capture and release) coated magnetic beads used as analyte cargos over droplets. Droplets ranging from 1 to 10 µL play the role of confined microreactors, allowing to realize several steps that involve advanced functions such as heating and mixing with organic solvents. A complex sample treatment protocol that has been feasible so far only in batchwise mode can now be converted into a novel microfluidic version. With this Lab-in-Droplet, we can enzymatically release and fluorescently label N-linked oligosaccharides from Human Immuglobulin G and then off-line analyze the labeled glycans by capillary electrophoresis with laser induced fluorescent detection. We demonstrated the superiority of this Lab-in-Droplet over the conventional batchwise protocol, with 10-fold less reagent consumption, 3-fold less time, and 2-fold improvement of glycan labeling yield, without degradation of glycan separation profile obtained by capillary electrophoresis. The platform with the developed droplet protocol was applied successfully for mapping N-linked glycans released from human sera, serving for diagnostic screening of congenital disorders of glycosylation.