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.

In this study, we report the development of a simple and cost-effective approach for fish sauce quality control. This is based on simultaneous determination of histamine and tyrosine in fish sauce using capillary electrophoresis (CE) coupled with contactless conductivity detection (C4D). Considerations for the use of purpose-made CE-C4D for food quality control in Vietnam are provided. The best detection limit (LOD) achieved was 5mg/kg for histamine and 6mg/kg for tyramine using the optimized background electrolyte (BGE) composed of 20mM (N-(2-Hydroxyethyl)piperazine-N’-(2-ethane-sulfonic acid]) (HEPES) / 10 mM L-Histidine (His), 5% methanol (MeOH) at pH 6.6. Excellent agreement between the results from CE-C4D and those from the confirmation method (HPLC-PDA for histamine and HPLC-FLD for tyramine) was achieved for different fish sauce samples, with their result deviations less than 10% for all tested compounds.

In this study, an affordable and versatile tool for screening illegal synthetic compounds in herbal-based products and nutrition supplements is proposed and discussed. The approach is based on capillary electrophoresis (CE) instrumentation, with the evolution from purpose-made to modular setups, coupled with capacitively coupled contactless conductivity detection (C4D). Viewpoints on the use of purpose-made CE-C4D towards modular systems for food quality control in Vietnam are provided. Our CE-C4D approach was applied to screen a number of various synthetic compounds illegally used in different herbal-based products and nutrient supplements, including i) determination of phenylpropanolamine, fenfluramine, sibutramine, and benzylsibutramine in weight-loss products, ii) analysis of propranolol, terazosin, and furosemide in products for hypertension treatment, iii) screening of sildenafil, homosildenafil, vardenafil, vardenafil acetyl, mirodenafil, hydroxy acetildenafil in nutrients for male sexual health and iv) identification of hypoglycemic agents (metformin, phenformin, glibenclamide, gliclazide) in diabetic treatment drugs. Good agreement between results obtained with our CE-C4D methods and those from the established confirmation method (LC-MS/MS) was achieved, with deviation less than 10 % for all categories of investigated compounds.

Background For healthcare applications of nanoparticles (NPs), their analysis and characterization are essential to obtain information on their heterogeneity regarding their size, shape, and surface chemistry or functionalization. Among the different analytical methods available, capillary electrophoresis (CE) is a well-established technique for such purpose. Nevertheless, CE approaches often suffer from unsatisfactory detection sensitivity when dealing with low NPs concentrations. In a related context, working with low NPs concentrations is essential in cellular engineering to minimize their cytotoxicity, which is notably caused by nanoparticle aggregation at high concentrations due to insufficient colloidal stability. Results We present in this study a novel principle of dual cycles of dual-stage electrokinetic preconcentration to drastically improve the detection sensitivity for characterization of core-shell magnetic nanoparticles (MNPs) by CE coupled with laser-induced fluorescent (LIF) detection. This approach is based on the combination and repetition of large volume sample stacking with electroosmotic pump (LVSEP) and transient isotachophoresis (tITP) using modulation of the electro-osmotic flow without recourse to any capillary coating. This dual cycle LVSEP-tITP approach allows enrichment of the MNPs having heterogeneous mobilities from a sample volume equivalent to 170 % of the total capillary volume. To demonstrate the significance of dual cycle LVSEP-tITP, the preconcentration principle was applied for CE-LIF of different populations of labelled core-shell MNPs functionalized with sulfobetaine having different sizes (40 and 125 nm) and forms (spherical and non-spherical), offering enrichment factors up to 110-fold compared to that obtained with CE-LIF without the preconcentration steps. Significance This study describes a new preconcentration concept for high-performance detection and electrokinetic fingerprinting of MNPs with heterogeneous subpopulations and mobilities. This will open the floor for characterization of MNPs in their suspension media after their multiple-step synthesis, providing guides for re-optimization of the synthesis process.

In this study, we investigated the behavior of extracellular vesicles (EVs), during capillary isoelectric focusing (cIEF). For that, we used different approaches, imaging cIEF with a whole-column imaging detection (WCID) and conventional cIEF as well as different detection methods (LIF after EV labelling, native fluorescence and UV). Our study reveals that EVs exhibit significant aggregation during their migration toward, and upon reaching, their isoelectric point (pI). By optimizing key parameters such as voltage and the addition of solubilizers, we successfully reduced this issue, particularly with bovine milk EVs. Our findings also showed distinct pI regions observed for EVs isolated from different sources: bovine milk EVs shows acidic pI characteristics (4.0-4.1), while pig and human plasma EVs exhibit more basic pI zones (4.7-4.9 and 5.8-6.7, respectively). The study was extended to cIEF coupled to laser induced fluorescence detection (LIF) using intra-vesicular CFDA-labeled EVs, to better understand their susceptibilities. Prolonged mobilization time due to long capillary lengths adversely affected EV's integrity in conventional cIEF. Our study reveals the necessity to specific cIEF optimization for each EV source due to variations in charge distribution and aggregation behavior across different pI regions. The use of a short capillary length (<10 cm), low electric field and solubilizers such as Tween-20 is recommended to preserve EVs integrity during cIEF-EV studies.

This study reports on the development of a novel modular microfluidic capillary electrophoresis (MMCE) platform with LED-based fluorescent (LEDIF) detection for automatic sampling and analysis purpose. The MMCELEDIF design is based on unprecedented 'plugging' hyphenation of various off-the-shelf parts available for microfluidics, optics and electrophoresis, allowing the users to construct their own analytical device without requirement of mechanical and electronic workshop facilities. The detection module relies on a LED source to generate a defined excitation wavelength, which can be focused on an optical window along the separation capillary to excite the separated target molecules. For automatic sampling and analysis, pneumatically driven modules were coupled with capillary electrophoresis (CE) to replace the conventional sequential injection analysis (SIA)-CE setup, allowing continuous flow circulation (to mimic a bio-fluid circulation) and pulseless sample delivery and injection for analysis. The MMCE-LEDIF platform was applied for separation and fluorescent detection of tailored and functionalized magnetic nanoparticles, as well as monitoring of their interaction with dopamine to evaluate their potential as drug delivery nanoplatforms. Such operation was carried out in a continuous microflow of artificial cerebrospinal fluid to investigate their behavior in a bio-fluid mimicking environment.

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.

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.

This work explores strategies for electrokinetic preconcentration of extracellular vesicles (EVs) that are potential source of biomarkers for different diseases. The first approach that led to successful preconcentration of EVs is based on large volume sample stacking (LVSS), allowing an enrichment factor of 7 for CE of EVs with long-end injection (using a capillary with an effective length of 50 cm). Attempts were also made to perform multiple cycles of LVSS, field amplified sample stacking (FASS) and field amplified sample injection (FASI), to improve EVs preconcentration performance. The focus was then put on development of capillary isotachophoresis under high ionic strengths (IS) for electrokinetic enrichment of slow migrating EVs having heterogeneous mobilities. This approach relies on the use of extremely high concentrations of the terminating electrolyte (TE) to slow down the mobility of TE co-ions, rendering them slower than those of EVs. The limit of detection for intact EVs using the developed ITP-UV method reached 8.3 × 108 EVs/mL, allowing an enrichment of 25 folds and a linear calibration up to 4 × 1010 EVs/mL. The ITP-UV and ITP-LIF approaches were applied to provide the electrokinetic signature of EVs of bovine milk and human plasma as well as to visualize more specifically intravesicular fluorescently labelled EVs. The investigation of these strategies shredded light into the challenges still encountered with electrokinetic preconcentration and separation of heterogeneous EVs sub-populations which are discussed herein based on our results and other attempts reported in the literature.

Sudan III, a commonly used dye in cosmetics, poses health risks due to its potential carcinogenicity and allergenic properties, necessitating rigorous monitoring of its illegal presence in consumer goods. In this study, a novel fluorometric assay employing Nitrogen-doped Carbon Quantum Dots coated with Molecularly Imprinted Polymers (NCQDs/SiO2@MIP) is presented to accurately and sensitively detect Sudan III. Unlike other majority analytes, Sudan III shows fluorescence enhancement upon binding with our NCQDs-MIPs. Under optimal conditions-pH 7.0, histidine buffer, 15 min incubation, and 500 mg/L NCQDs/SiO2@MIP-the method achieves a low 1.4 nM (0.49 ppb) detection limit and a broad linear range (5-800 nM). With precision (RSD < 5 %) and selectivity surpassing Sudan dyes and common cosmetic ingredients, the method's reliability is evident. Cross-validation against LC-MS/MS confirms the accuracy of Sudan III quantification in real cosmetic samples. This NCQDs/SiO2@MIP fluorometric sensor holds promise for rapid on-site detection of Sudan III and other contaminants in cosmetics, offering superior sensitivity, selectivity, and minimal sample preparation requirements.

In this study, we report the development of versatile and cost-effective analytical approaches for analysis and quality control of different nutraceutical and tonic products, using capillary electrophoresis (CE) coupled with contactless conductivity detection ((CD)-D-4). Insights into CE-(CD)-D-4 instrumental designs that are adapted to the context in developing countries are provided. A highlight was made for quality control of vitamins, glucosamine and some minerals in different nutraceutical formulations collected from pharmacies in Vietnam. The lowest detection limit (LOD) achieved with the developed methods was 0.1 mg/L for vitamins, using the optimized background electrolyte (BGE) composed of 12 mM arginine (Arg)/acetic acid (AcOH) at pH 7.5 with 10 % acetonitrile (ACN). For magnesium and B6, the optimized CE-(CD)-D-4 conditions with the BGE composed of 10 mM Arg/AcOH (pH 5) with 20 % ACN allows its detection down to 0.05 mg/L. For glucosamine and calcium, the best performance with LOD of 0.05 mg/L was achieved with BGE composed of 10 mM Tris/AcOH (pH 5). Good agreement between results from CE-(CD)-D-4 and the confirmation methods was achieved for all investigated compounds, with their result deviations less than 15 %.

This study reports on the development of a new concept of on-line dual preconcentration stages for capillary electrophoresis (CE), in which two completely different preconcentration approaches can be realized in the same capillary. In the first stage, a dynamic magneto-extraction of target analytes on circulating magnetic beads is implemented within the capillary. In the second one, electrokinetic preconcentration of eluted analytes via large volume sample stacking is carried out to focus them into a nano band, prior to CE separation of enriched analytes. To implement the dual-stage preconcentration operation, a purpose-made instrument was designed, combining electrophoretic and microfluidic modules to allow precise control of the movement of magnetic beads and analyte's flow. The potential of this new enrichment principle and its associated instrument was demonstrated for CE separation with light-emitting-diode-induced fluorescent (LEDIF) detection of target double-stranded DNA (ds-DNA). The workflow consists of purification and preconcentration of a target DNA fragment (300 bp) on negatively charged magnetic beads, followed by in-capillary elution and fluorescent labelling of the enriched DNA. Large volume sample stacking of the DNA eluent was then triggered to further preconcentrate the labelled DNA before its analysis by CE-LEDIF. An enrichment factor of 125 was achieved for the target DNA fragment. With our new approach, dual-stage sample pretreatment and CE separation can now be performed in-capillary without any mismatch of working volumes, nor any waste of pretreated samples.

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 (30-10 0 0 nm). Here, we showed that TDA, conducted in uncoated fused silica capillaries (50 & mu;m 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 x 10 11 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.& COPY; 2023 Elsevier B.V. All rights reserved.

It is reported herein a new approach to study the orientation and density of mouse antibody grafting on magnetic beads, serving for immunoassays and biosensors with fluorescent detection of biomolecules. This approach is based on selective enzymatic digestion of target grafted antibodies at a specific site below the hinge position to provide F(ab ')2 and Fc fragments, followed by separation and determination of these fragments with size exclusion chromatography (SEC) coupled with fluorescence detection (FLD). The developed method was applied for evaluation of immunoglobulin (IgG2a) grafting capacity on three different biofunctionalized magnetic beads (i.e., Tosyl-activated, carboxylic, protein G). Tosyl-activated and protein G beads at different optimal grafting IgG: bead ratios (i.e., 110 mu g: 1000 mu g and 240 mu g: 1000 mu g, respectively) exhibited superior grafting capacity than carboxylic counterparts. Under the optimized conditions, more than 70 % of antibodies were grafted on tosyl-activated and protein G beads in the right orientation. This approach was then demonstrated with different commercially available antibodies specific to amyloid-beta peptide 1-42 (A beta 1-42) for magneto-immunoassays and fluorescent detection of this peptide that is an established biomarker for molecular diagnosis of Alzheimer's disease.

The energy sector is an intrinsically dynamic and complex system, and therefore its behaviour is not solely controlled by constituent components. Rather, it is a consequence of dynamic interactions among them. To properly manage such a system in a sustainable manner, it is necessary to understand the underlying dynamics of component interactions. Despite this, the interconnections between components of the energy sector in research and policy have received little attention. Here, we outline crucial limitations of previous efforts and emphasize the importance of using systems thinking in addressing the energy sector's sustainability challenges. We demonstrate this by a case study of the Australian energy sector, which has experienced emerging sustainability issues. Research findings show that current policies promoting energy development in the country are likely to be 'fixes that fail' that ultimately undermine sustainability. To achieve in building a sustainable energy sector, the policy must focus on implementing long-term solutions and avoid short-term quick fixes.

The cytokine interleukin 6 (IL-6) is involved in the pathogenesis of different inflammatory diseases, including cancer, and its monitoring could help diagnosis, prognosis of relapse-free survival and recurrence. Here, we report an innovative microfluidic approach that uses the fluidization of magnetic beads to specifically extract, preconcentrate and fluorescently detect IL-6 directly on-chip. We assess how the physical properties of the beads can be tuned to improve assay performance by enhancing mass transport, reduce non-specific binding and multiply the detection signal threefold by transitioning between packed and fluidization states. With the integration of a full ELISA protocol in a single microfluidic chamber, we show a twofold reduction in LOD compared to conventional methods along with a large dynamic range (10 pg/mL to 2 ng/mL). We additionally demonstrate its application to IL-6 detection in undiluted serum samples.

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.

In this study, we present a new approach for in-capillary fluorescent labeling of N-glycans prior to their analysis with CE coupled with laser-induced fluorescent detection. This integrated approach allows using a CE capillary as a microreactor to perform several steps required for labeling glycans with 8-aminopyrene-1,3,6 trisulfonic acid and at the same time as a separation channel for CE of fluorescently labeled glycans. This could be achieved through careful optimization of all different steps, including sequential injections of fluorescent dye and glycan plugs, mixing by transverse diffusion of laminar flow profiles, incubation in a thermostatic zone, and finally separation and detection with CE. Such a complex sample treatment protocol for glycan labeling that is feasible thus far only in batchwise mode can now be converted into an automated and integrated protocol. Our approach was applied successfully to analyze fluorescently labeled N-linked oligosaccharides released from human immunoglobulin G and rituximab, a monoclonal antibody used for cancer treatment. We demonstrated the superiority of this in-capillary approach over the conventional in-tube protocol, with fourfold less reagent consumption and full automation without remarkable degradation of the glycan separation profile obtained by capillary electrophoresis.

Vitamins and minerals are usually incorporated in pharmaceutical and nutraceutical products, but a simple, rapid, and inexpensive analytical method for their simultaneous determination is still lacking. In this study, we developed a quantification method for pyridoxine (vitamin B6) and magnesium (Mg) by using purpose-made capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C4D) instrument. Main analytical conditions include: fused silica capillary (total length 55 cm, effective length 40 cm, inner diameter 50 mu m); background electrolyte consisted of 10 mM L-arginine/acetic acid (pH 5) with 20% acetonitrile; separation voltage + 20 kV; hydrodynamic injection (siphoning at 20 cm in 25 s). Detection limits of vitamin B6 and Mg were 1 and 0.1 mg/L, respectively. Good linearity (R2 > 0.999) was observed for vitamin B6 and Mg calibration curves over concentration ranges of 3-100 and 0.3-200 mg/L, respectively. The method was applied to analyze vitamin B6 and Mg in several pharmaceutical and nutraceutical samples. The analytical results obtained by our method were in good agreement with reference methods (i.e., HPLC for vitamin B6 and ICP-OES for Mg). High-efficient and low-cost CE-C4D method can accordingly serve as a promising tool for concurrent analysis of inorganic and organic species in pharmaceutical and nutraceutical analysis.

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.