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.

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.

On-line dual-stage enrichment via magneto-extraction and electrokinetic preconcentration: A new concept and instrumentation for capillary electrophoresis.

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.

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.

In capillary labeling and online electrophoretic separation of N-glycans from glycoproteins.

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.

Modular microfluidic system for on-chip extraction, preconcentration and detection of the cytokine biomarker IL-6 in biofluid.

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.

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.

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.

Analytical methods of antibody surface coverage and orientation on bio-functionalized magnetic beads: application to immunocapture of TNF-α.

The use of magnetic beads bio-functionalized by antibodies (Ab) is constantly increasing with a wide range of biomedical applications. However, despite an urgent need for current methods to monitor Ab's grafting process and orientation, existing methods are still either cumbersome and/or limited. In this work, we propose a new simple and rapid analytical approach to evaluate antibody orientation and density on magnetic beads. This approach relies on the cleavage by IdeS, a highly specific protease for human immunoglobulin G (hIgG), of immobilized antibodies. The F(ab)2 and Fc fragments could be then accurately quantified by size exclusion chromatography (SEC)-coupled to fluorescent detection (FLD), and the ratio of these fragments was used to give insight on the IgG orientation at the bead surface. Four different commercially available magnetic beads, bearing carboxyl groups, tosyl groups, streptavidin, or protein G on their surface have been used in this study. Results obtained showed that this approach ensures reliable information on hIgG orientation and bead surface coverage. Protein G magnetic beads demonstrated an optimal orientation of antibodies for antigen capture (75% of accessible F(ab)2 fragment) compared to tosylactivated, carboxylated, and streptavidin ones. Capture efficiency of the different functionalized beads towards human TNF-α immunocapture, a biomarker of inflammation, has been also compared. Protein G beads provided a more efficient capture compared to other beads. In the future, this approach could be applied to any type of surface and beads to assess hIgG coverage and orientation after any type of immobilization. A rapid and simple approach to evaluate orientation and density of antibodies immobilized on magnetic beads.

Electroosmotic flow modulation for improved electrokinetic preconcentration: Application to capillary electrophoresis of fluorescent magnetic nanoparticles.

It is reported in this study a new approach for modulation and even suppression of the electroosmotic flow (EOF) to achieve better electrokinetic preconcentration in capillary electrophoresis. This is based on the augmentation of the buffer's concentrations to very high levels (more than a thousand of mM) without recourse to any dynamic/permanent coating nor viscous gel. The use of large weakly charged molecules as background electrolyte's constituents allows working at extreme concentration ranges without penalty of high electric currents and Joule heating. By this way, the electroosmotic mobility could be modulated over a wide range (2-60 × 10-5 cm2 V-1 s-1 under alkaline conditions), and suppressed to levels equivalent to those obtained with several neutral coatings. The highest buffer concentrations, and the lowest EOF magnitudes, accordingly, were achieved with diethanolamine/3-(Cyclohexylamino)-1-propanesulfonic acid (ionic strength (IS) of 250 mM, pH 9.5), Tris(hydroxymethyl)aminomethane (Tris)/2-(Cyclohexylamino)ethanesulfonic acid (CHES) (IS of 280 mM, pH 8.7) and triethanolamine/2-(Cyclohexylamino)ethanesulfonic acid (IS of 250 mM, pH 8.5). For demonstration, this new approach was applied for sensitive determination of core-shell magnetic nanoparticles (CSMNPs) having high potential for healthcare applications such as imaging agents for diagnostics and controllable cargos for nanomedicine. Different profiles were achieved for purpose-made and commercial magnetic nanoparticles using CE coupled with light-emitting-diode induced fluorescence (LEDIF) detection. The best performance for EOF-assisted preconcentration and CE-LEDIF of CSMNPs was achieved with these nanoparticles prepared in TRIS/CHES (IS 10 mM, pH 8.4) for preconcentration, and separation under BGE of TRIS/CHES (IS 100 mM, pH 8.4). Compared to the conventional capillary electrophoresis (CE-UV) method for characterization of magnetic nanoparticles, our proposed approach with fluorescent detection and EOF-assisted preconcentration offers almost 350-fold sensitivity improvement. Furthermore, our scheme can be used for monitoring the interaction between CSMNPs and target pharmaceutical molecules, serving for drug delivery development. A preliminary study with two antibiotics using this approach revealed that kanamycin interacts better with the target nanoparticles than amikacin.

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.

Recent Electrokinetic and Microfluidic Strategies for Detection of Amyloid Beta Peptide Biomarkers: Towards Molecular Diagnosis of Alzheimer's Disease.

Among all neurodegenerative diseases, Alzheimer's Disease (AD) is the most prevalent worldwide, with a huge burden to the society and no efficient AD treatment so far. Continued efforts have been being made towards early and powerful diagnosis of AD, in the hope for a successful set of clinical trials and subsequently AD curative treatment. Towards this aim, detection and quantification of amyloid beta (Aß) peptides in cerebrospinal fluid (CSF) and other biofluids, which are established and validated biomarkers for AD, have drawn attention of the scientific community and industry over almost two decades. In this work, an overview on our major contributions over 15 years to develop different electrokinetic and microfluidic strategies for Aß peptides detection and quantification is reported. Accordingly, discussions and viewpoints on instrumental and methodological developments for microscale electrophoresis, microfluidic designs and immuno-enrichment / assays on magnetic beads in microchannels for tracing Aß peptides in CSF are given in this review.

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.

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.

Low-cost and versatile analytical tool with purpose-made capillary electrophoresis coupled to contactless conductivity detection: Application to antibiotics quality control in Vietnam.

In this study, the development of our purpose-made capacitively coupled contactless conductivity detection (C4 D) for CE is reported. These systems have been employed as a simple, versatile, and cost-effective analytical tool. CE-C4 D devices, whose principle is based on the control of the ion movements under an electrical field, can be constructed even with a modest financial budget and limited infrastructure. A featured application was developed for quality control of antimicrobial drugs using CE-C4 D, with most recent work on determination of aminoglycoside and glycopeptide antibiotics being communicated. For aminoglycosides, the development of CE-C4 D methods was adapted to two categories. The first one includes drugs (liquid or powder form) for intravenous injection, containing either amikacin, streptomycin, kanamycin A, or kanamycin B. The second one covers drugs for eye drops (liquid or ointment form), containing either neomycin, tobramycin, or polymyxin. The CE-C4 D method development was also made for determination of some popular glycopeptide antibiotics in Vietnam, including vancomycin and teicoplanin. The best detection limit achieved using the developed CE-C4 D methods was 0.5 mg/L. Good agreement between results from CE-C4 D and the confirmation method (HPLC- Photometric Diode Array ) was achieved, with their result deviations less than 8% and 13% for aminoglycoside and glycopeptide antibiotics, respectively.

CDG biochemical screening: Where do we stand?

BACKGROUND: Glycosylation is one of the most complex post-translational modifications of proteins and lipids, notably requiring many glycosyltransferases, glycosidases and sugar transporters encoded by about 1-2% of all human genes. Deleterious variants in any of them may result in improper protein or lipid glycosylation, thus yielding the so-called 'congenital disorders of glycosylation' or CDG. SCOPE OF REVIEW: We first review the current state of knowledge on the common blood and cellular glycoproteins used in the biochemical screening of CDG, as well as the emerging ones for an improved diagnosis. We then provide an overview of the current state-of-the-art methodologies ranging from gel electrophoresis to mass spectrometry to measure improper glycosylation. Finally, we discuss how additional tools such as metabolomics and microfluidics can be added to the current toolbox to better diagnose and delineate CDG. MAJOR CONCLUSIONS: Combining several biochemical indicators and related methods is often required to cope with the large clinical heterogeneity of CDG and establish a definitive diagnosis. GENERAL SIGNIFICANCE: This review aims to critically present current available CDG biochemical biomarkers and dedicated methods in the context of highly diverse glycosylation pathways and related inherited diseases.

Determination of carbapenem antibiotics using a purpose-made capillary electrophoresis instrument with contactless conductivity detection.

In this study, the employment of a purpose-made capillary electrophoresis (CE) instrument with capacitively coupled contactless conductivity detection (C4D) as a simple and cost-effective approach for simultaneous determination of different carbapenem antibiotics is reported. The developed CE-C4D approach was for the first time applied for quality control of various pharmaceutical formulations in Vietnam, as well as for therapeutic monitoring of these antibiotics in plasma samples from patients under intensive care. Four of the most popular carbapenems in Vietnam, doripenem, meropenem, imipenem and ertapenem, were determined using an electrolyte composed of 10 mM Tris adjusted to pH 8.0 with acetic acid. The best detection limits achieved using the developed CE-C4D method were 0.36 mg/L and 0.45 mg/L for pharmaceutical and plasma samples, respectively. Good agreement between results from CE-C4D and the confirmation method (HPLC-PDA) was achieved, with a coefficient of determination (r2) for the two pairs of data of 0.9967.

Cost-effective capillary electrophoresis with contactless conductivity detection for quality control of beta-lactam antibiotics.

A simple and inexpensive approach for determination of various antimicrobial drugs using a purpose-made compact capillary electrophoresis (CE) instrument with capacitively coupled contactless conductivity detection (C4D) is reported. The objective of the work is to propose an affordable and easily-implemented tool for quality control and detection of counterfeiting of antibiotic formulations in resource constrained developing countries. The design of the purpose-made CE-C4D system was improved according to the feedback from over 10 years of use of our previous instrument. CE-C4D methods were for the first time developed to analyze ß-lactam-based antibiotics commonly used in Vietnam, including single- ß-lactam antibiotics (i.e. Cephalexin, Cefotaxime Sodium, Cefixime and Sulbactam) as well as ß-lactams co-formulated with Sulbactam (i.e. Amoxicillin, Ampicillin, Cefoperazone and Sulbactam). Single ß-lactam antibiotics were analyzed using a background electrolyte (BGE) composed of Tris/Ace (10 mM, pH 7.8) whereas ß-lactam - Sulbactam combinations were simultaneously separated using a BGE containing Tris/Ace (10 mM, pH 7.5). The best achieved detection limits were 2.0 mg/L and 1.0 mg/L for these two groups, respectively. Good agreement between results obtained from CE-C4D and standard confirmation methods (LCMS) was achieved, with a coefficient of determination, r2, of 0.9991. The applicability of the developed CE-C4D method was demonstrated for quality control of 24 ß-lactam-based antimicrobial drugs available in Vietnam.

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.