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.

A review on hyphenation of droplet microfluidics to separation techniques: From instrumental conception to analytical applications for limited sample volumes.

In this study, we review various strategies to couple sample processing in microfluidic droplets with different separation techniques, including liquid chromatography, mass spectrometry, and capillary electrophoresis. Separation techniques interfaced with droplet microfluidics represent an emerging trend in analytical chemistry, in which micro to femtoliter droplets serve as microreactors, a bridge between analytical modules, as well as carriers of target analytes between sample treatment and separation/detection steps. This allows to overcome the hurdles encountered in separation science, notably the low degree of module integration, working volume incompatibility, and cross contamination between different operational stages. For this droplet-separation interfacing purpose, this review covers different instrumental designs from all works on this topic up to May 2023, together with our viewpoints on respective advantages and considerations. Demonstration and performance of droplet-interfaced separation strategies for limited sample volumes are also discussed.

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.

High sensitivity capillary electrophoresis with fluorescent detection for glycan mapping.

We present in this study a novel strategy to drastically improve the detection sensitivity and peak capacity for capillary electrophoresis with laser induced fluorescent detection (CE-LIF) of glucose oligomers and released glycans. This is based on a new approach exploiting a polymer-free background electrolyte (BGE) for CE-LIF of glycans. The best performance in terms of sample stacking and suppression of electroosmotic flow (EOF) was found for a BGE composed of triethanolamine/citric acid and triethanolamine/acetic acid at elevated ionic strengths (IS up to 200 mM). Compared to the conventional protocols for CE-LIF of glucose-oligosaccharides and released glycans, our polymer-free strategy offered up to 5-fold improvement of detection sensitivity and visualization of higher degree of polymerization (DP) of glucose oligomers (18 vs 15). To further improve the detection sensitivity, a new electrokinetic preconcentration strategy via large volume sample stacking with electroosmotic modulation without having recourse to neutrally coated capillaries is proposed, offering a 200-fold signal enhancement. This approach is based on variation of the buffer's IS, rather than pH adjustment as in conventional methods, for EOF modulation or quasi-total reduction. This strategy allows selecting with high flexibility the best pH conditions to perform efficient preconcentration and separation. The new approach was demonstrated to be applicable for the analysis of N-linked oligosaccharides released from a model glycoprotein (Human Immunoglobulin G) and applied to map N-glycans from human serum for congenital disorders of glycosylation (CDG) diagnosis.

Droplet-interfacing strategies in microscale electrophoresis for sample treatment, separation and quantification: A review.

In this study, for the first time we report on a comprehensive overview of different strategies to hyphenate droplet-based sample handling and preparation with electrophoretic separation in different formats (i.e. microchip and capillary electrophoresis). Droplet-interfaced electrophoresis is an emerging technique in which micro/nanometric droplets are used as a bridge and carrier of target analytes between sample treatment and electrokinetic separation steps, thus being expected to overcome the challenges of working dimension mismatch and low degree of module integration. This review covers all works on this topic from 2006 (the year of the first communication) up to 2020, with focus being given to three principal interfacing strategies, including droplets in immiscible phases, digital microfluidics with electrowetting-on-dielectric principle and inkjet droplet generation. Different instrumental developments for such purpose, the viewpoints on pros and cons of these designs as well as application demonstrations of droplet-interfaced electrokinetic strategies are discussed.

Modular instrumentation for capillary electrophoresis with laser induced fluorescence detection using plug-and-play microfluidic, electrophoretic and optic modules.

This study reports on the development of a novel instrument for capillary electrophoresis (CE) coupled with laser induced fluorescence (LIF) detection that is inspired by the Lego-toy concept. The Lego CE-LIF design is an evolution of purpose-made CE instrumentation, allowing the users to construct their own analytical device with a high degree of standardization (i.e. a "standard" setup) without requirement of mechanical and electronic workshop facilities. To allow instrument reproduction outside the original fabrication laboratory, which is not trivial for in-house-built CE systems, the new design is based on unprecedent 'plugging' hyphenation of various off-the-shelf parts available for microfluidics, optics and electrophoresis. To render the operation with Lego CE-LIF optimal, we developed a new background electrolyte (BGE), using for the first time extremely high concentrations of zwitterionic and large weakly charged species for much improvement of detection sensitivity. The Lego CE-LIF was demonstrated for separation and detection of oligosaccharides labelled with 8-aminopyrene-1,3,6-trisulfonic acid (APTS). The new gel-free BGE for oligosaccharide analysis also allowed simplification of the conventional CE-LIF protocol used with commercial instruments while keeping satisfactory separation performances. Furthermore, the new BGE is fully compatible with a non-thermostatted Lego CE instrument thanks to low current and therefore low heat generation under application of a high voltage.