Fast, simple and calibration-free size characterization and quality control of extracellular vesicles using capillary Taylor dispersion analysis.

This study reports the development of a Taylor Dispersion Analysis (TDA) method for the size characterization of Extracellular Vesicles (EVs), which are highly heterogeneous nanoscale cell-derived vesicles (30-1000 nm). Here, we showed that TDA, conducted in uncoated fused silica capillaries (50 µm i.d.) using a conventional Capillary Electrophoresis instrument, is able to provide absolute sizing (requiring no calibration) of bovine milk-derived EVs in a small sample volume (∼ 7 nL) and over their entire size range, even the smallest ones (< 70 nm) not accessible via other techniques that provide nanoparticle sizing in suspension. TDA size measurements were repeatable (RSD < 10%) and the average EV sizes were found in the range of 120-210 nm, in very good agreement with those measured with Nanoparticle Tracking Analysis, commonly used for EV characterization. TDA allowed quantitative estimation of EVs for concentrations ≥ 2 × 1011 EVs/mL. Furthermore, TDA was able to detect minor changes in EV size (i.e. by ∼25 nm upon interaction with specific anti-CD9 antibodies of ∼150 kDa), and to highlight the impact of extraction methods (i.e. milk pretreatment: freezing, acid precipitation or centrifugation; the type of size-exclusion chromatography column) and of fluorescent labeling (i.e. intravesicular or surface labeling) on the isolated EV population size. In parallel to EV sizing, TDA allowed to detect molecular contaminants (average sizes ∼1-13 nm) present within the sample, rendering this method a valuable tool to assess the quality and quantity of EV isolates.

Development of a microfluidic droplet platform with an antibody-free magnetic-bead-based strategy for high through-put and efficient EVs isolation.

In this study, we present a novel microfluidic droplet-based strategy for high performance isolation of extracellular vesicles (EVs). For EVs capture and release, a magnetic bead-based approach without having recourse to any antibody was optimized in batch and then adapted to the microfluidic droplet system. This antibody-free capture approach relies on the presence of a water-excluding polymer, polyethylene glycol (PEG), to precipitate EVs on the surface of negatively charged magnetic beads. We significantly improved the reproducibility of EV recovery and avoided positive false bias by including a washing step and optimizing the protocol. Well-characterized EV standards derived from pre-purified bovine milk were used for EVs isolation performance evaluation. An EVs recovery of up to 25% estimated with nanoparticle tracking analysis (NTA) was achieved for this batchwise PEG-based approach. The confirmation of isolated EVs identity was also made with our recently developed method using capillary electrophoresis (CE) coupled with laser-induced fluorescent (LIF) detection. In parallel, a purpose-made droplet platform working with magnetic tweezers was developed for translation of this PEG-based method into a droplet microfluidic protocol to further improve the performance in terms of EVs capture efficiency and high throughput. The droplet-based protocol offers a significant improvement of recovery rate (up to 50%) while reducing sample and reagent volumes (by more than 10 folds) and operation time (by 3 folds) compared to the batch-wise mode.

Electrokinetic characterization of extracellular vesicles with capillary electrophoresis: A new tool for their identification and quantification.

This work reports on the development of the first capillary electrophoresis methodology for the elucidation of extracellular vesicles' (EVs) electrokinetic distributions. The approach is based on capillary electrophoresis coupled with laser-induced fluorescent (LIF) detection for the identification and quantification of EVs after their isolation. Sensitive detection of these nanometric entities was possible thanks to an 'inorganic-species-free' background electrolyte. This electrolyte was made up of weakly charged molecules at very high concentrations to stabilize EVs, and an intra-membrane labelling approach was used to prevent EV morphology modification. The limit of detection for EVs achieved using the developed CE-LIF method reached 8 × 109 EV/mL, whereas the calibration curve was acquired from 1.22 × 1010 to 1.20 × 1011 EV/mL. The CE-LIF approach was applied to provide the electrokinetic distributions of various EVs of animal and human origins, and visualize different EV subpopulations from our recently developed high-yield EV isolation method.

A fresh look into background electrolyte selection for capillary electrophoresis-laser induced fluorescence of peptides and proteins.

This study reports a reinvestigation of background electrolyte selection strategy for performance improvement in CE-LIF of peptides and proteins. This strategy is based on the employment of high concentrations of organic species in BGE possessing high buffer capacity and low specific conductivity in order to ensure excellent stacking preconcentration and separation resolution of fluorescently tagged peptides and proteins. Unlike universal UV detection, the use of such BGEs at high concentrations does not lead to degradation of LIF detection signals at the working excitation and emission wavelengths. At the same buffer ionic strength, pH and electric field, an "inorganic-species-free" BGE (or ISF BGE) for CE-LIF of fluorescently labeled beta amyloid peptide Aß 1-42 (a model analyte) offered a signal intensity and peak efficiency at least three-times higher than those obtained with a conventional BGE normally used for CE-LIF, while producing an electric current twice lower. Good peak performance (in terms of height and shape) was maintained when using ISF BGEs even with samples prepared in high-conductivity phosphate buffer saline matrix. The advantageous features of such BGEs used at high concentrations over conventional ones in terms of high separation resolution, improved signal intensities, tuning of EOF magnitudes and minimization of protein adsorption on an uncoated fused silica capillary are demonstrated using Alexa-488-labelled trypsin inhibitor. Such BGE selection approach was applied for investigation of separation performance for CE-LIF of ovalbumin labelled with different fluorophores.

A capillary zone electrophoresis method for detection of Apolipoprotein C-III glycoforms and other related artifactually modified species.

ApolipoproteinC-III (ApoC-III) is a human plasma glycoprotein whose O-glycosylation can be altered as a result of congenital disorders of glycosylation (CDG). ApoC-III exhibits three major glycoforms whose relative quantification is of utmost importance for the diagnosis of CDG patients. Considering the very close structures of these glycoforms and their tendency to adsorb on the capillary, a thorough optimization of capillary electrophoresis (CE) parameters including preconditioning and in-between rinsing procedures was required to efficiently separate all the ApoC-III glycoforms. Permanent coatings did not contribute to high resolution separations. A fast and reliable method based on a bare-silica capillary combining the effect of urea and diamine additives allowed to separate up to six different ApoC-III forms. We demonstrated by a combination of MALDI-TOF mass spectrometry (MS) analyses and CE of intact and neuraminidase-treated samples that this method well resolved glycoforms differing not only by their sialylation degree but also by carbamylation state, an undesired chemical modification of primary amines. This method allowed to demonstrate the carbamylation of ApoC-III glycoforms for the first time. Our CZE method proved robust and accurate with excellent intermediate precision regarding migration times (RSDs < 0.7%) while RSDs for peak areas were less than 5%. Finally, the quality of three distinct batches of commercial ApoC-III obtained from different suppliers was assessed and compared. Quite similar but highly structurally heterogeneous ApoC-III profiles were observed for these samples.