Atypical applications of transverse diffusion of laminar flow profiles methodology for in-capillary reactions in capillary electrophoresis.

Capillary electrophoresis (CE) is a powerful separation technique offering quick and efficient analyses in various fields of bioanalytical chemistry. It is characterized by many well-known advantages, but one, which is perhaps the most important for this application field, is somewhat overlooked. It is the possibility to perform chemical and biochemical reactions at the nL scale inside the separation capillary.

Contactless conductivity detector as a tool for improving universality and sensitivity of capillary electrophoresis-frontal analysis: Proof of concept.

Drug binding to plasma proteins influences processes such as liberation, adsorption, disposition, metabolism, and elimination of drugs, which are thus one of the key steps of a new drug development. As a result, the characterization of drug-protein interactions is an essential part of these time- and money-consuming processes. It is important to determine not only the binding strength and the stoichiometry of interaction, but also the binding site of a drug on a protein molecule, because two drugs with the same binding site can mutually affect free drug concentration.

Gene expression and metabolic activity of Streptococcus mutans during exposure to dietary carbohydrates glucose, sucrose, lactose, and xylitol.

Recent RNA sequencing studies have given us a deeper insight into the cariogenic impact of carbohydrate sources in the bacterium Streptococcus mutans, the principal microbial agent in dental caries etiopathogenesis. The process of dental caries development is facilitated by the ability of this bacterium to ferment some carbohydrates into organic acids contributing to a pH decrease in the oral cavity and the demineralization of the hard tissues of the tooth. Furthermore, in dental caries progression, biofilm formation, which starts and ends with free planktonic cells, plays an important role and has several unique properties called virulence factors.

Sensitivity enhancement of capillary electrophoresis-frontal analysis-based method for characterization of drug-protein interactions using on-line sample preconcentration.

Capillary electrophoresis-frontal analysis is one of the most frequently used approaches for the study of plasma protein-drug interactions as a substantial part of new drug development. However, the capillary electrophoresis-frontal analysis typically combined with ultraviolet-visible detection suffers from insufficient concentration sensitivity, particularly for substances with limited solubility and low molar absorption coefficient. The sensitivity problem has been solved in this work by its combination with an on-line sample preconcentration.

Studying molecular interactions via capillary electrophoresis and microscale thermophoresis: A review.

The process of choosing the most proper technique for studying the molecular interactions is based on critical factors such as instrumentation complexity, automation, experimental procedures, analysis time, consumables, and cost-value. This review has tracked the use of affinity capillary electrophoresis (ACE) and microscale thermophoresis (MST) techniques in the evaluation of molecular binding among different molecules during the 5 years 2016-2021. ACE has proved to be an attractive technique for biomolecular characterization with high resolution efficiency where small variations in several controlling factors can much improve such efficiency compared to other analytical techniques.

Comparison of mobility shift affinity capillary electrophoresis and capillary electrophoresis frontal analysis for binding constant determination between human serum albumin and small drugs.

In this study, two capillary electrophoresis-based ligand binding assays, namely, mobility shift affinity capillary electrophoresis (ms-ACE) and capillary electrophoresis-frontal analysis (CE-FA), were applied to determine binding parameters of human serum albumin toward small drugs under similar experimental conditions. The substances S-amlodipine (S-AML), lidocaine (LDC), l-tryptophan (l-TRP), carbamazepine (CBZ), ibuprofen (IBU), and R-verapamil (R-VPM) were used as the main binding partners. The scope of this comparative study was to estimate and compare both the assays in terms of their primary measure's precision and the reproducibility of the derived binding parameters.

Extending the application potential of capillary electrophoresis/frontal analysis for drug-plasma protein studies by combining it with mass spectrometry detection.

CE/frontal analysis (CE/FA) is probably one of the most frequently used modes of CE for studying affinity interactions. It is typically performed with classic UV-Vis detection that suffers from low concentration sensitivity. To overcome this limitation, the applicability of CE/FA in combination with ESI-MS detection for the investigation of drug-HSA interactions was demonstrated.

Capillary electrophoresis-mass spectrometry as a tool for the noninvasive target metabolomic analysis of underivatized amino acids for evaluating embryo viability in assisted reproduction.

Monitoring metabolite uptake and excretion in the culture medium is a noninvasive technique that is used for the metabolic study of cleaving embryos after in vitro fertilization. Low sample consumption, the versatility of the detection, and optimal sensitivity and selectivity are essential elements for extracellular metabolome analyses, and can be conveniently achieved by combining CE with mass spectrometric detection. This paper reports a method for amino acid determination in a limited volume sample (8 μL) of spent culture media collected after the cultivation of in vitro fertilized embryos.

Non-Invasive Human Embryo Metabolic Assessment as a Developmental Criterion.

The selection of a highly-viable single embryo in assisted reproductive technology requires an acceptable predictive method in order to reduce the multiple pregnancy rate and increase the success rate. In this study, the metabolomic profiling of growing and impaired embryos was assessed on the fifth day of fertilization using capillary electrophoresis in order to find a relationship between the profiling and embryo development, and then to provide a mechanistic insight into the appearance/depletion of the metabolites. This unique qualitative technique exhibited the appearance of most non-essential amino acids and lactate, and depleting the serine, alanyl-glutamine and pyruvate in such a manner that the embryos impaired in their development secreted a considerably higher level of lactate and consumed a significantly higher amount of alanyl-glutamine.

Toward an automated workflow for the study of plasma protein-drug interactions based on capillary electrophoresis-frontal analysis combined with in-capillary mixing of interacting partners.

Capillary electrophoresis-frontal analysis (CE-FA) together with mobility shift affinity CE is the most frequently used mode of affinity CE for a study of plasma protein-drug interactions, which is a substantial part of the early stage of drug discovery. Whereas in the classic CE-FA setup the sample is prepared by off-line mixing of the interaction partners in the sample vial outside the CE instrument and after a short incubation period loaded into the capillary and analysed, in this work a new methodological approach has been developed that combines CE-FA with the mixing of interacting partners directly inside the capillary. This combination gives rise to a fully automated and versatile methodology for the characterization of these binding interactions besides a substantial reduction in the amounts of sample compounds used.

Applicability of capillary electrophoresis-frontal analysis for displacement studies: Effect of several drugs on l-tryptophan and lidocaine binding to human serum albumin.

The effective concentration of a drug in the blood, i.e. the concentration of a free drug in the blood, is influenced by the strength of drug binding onto plasma proteins.

Metabolic Activity of Human Embryos after Thawing Differs in Atmosphere with Different Oxygen Concentrations.

The vitrification of human embryos is more and more frequently being utilized as a method of assisted reproduction. For this technique, gentle treatment of the embryos after thawing is crucial. In this study, the balance of amino acids released to/consumed from the cultivation media surrounding the warmed embryos was observed in the context of a cultivation environment, which was with the atmospheric oxygen concentration ≈20% or with a regulated oxygen level-hysiological (5%).

Aldehyde oxidase assay by capillary electrophoresis: From off-line, online up to immobilized enzyme reactor.

Capillary electrophoresis is a modern separation technique characterized by many benefits, which qualify it also for enzyme assays and the study of enzyme kinetics during drug development. Homogeneous or heterogeneous approaches can be followed for the enzymatic incubation. In this study, an immobilization procedure of aldehyde oxidase on magnetic particles was developed considering their integration with capillary electrophoresis.

Homocyclic o-dicarboxaldehydes: Derivatization reagents for sensitive analysis of amino acids and related compounds by capillary and microchip electrophoresis.

Amino acids are essential compounds for living organisms, and their determination in biological fluids is crucial for the clinical analysis and diagnosis of many diseases. However, the detection of most amino acids is hindered by the lack of a strong chromophore/fluorophore or electrochemically active group in their chemical structures. The highly sensitive determination of amino acids often requires derivatization.

Capillary electrophoresis-based immunoassay and aptamer assay: A review.

Over the last two decades, the group of techniques called affinity probe CE has been widely used for the detection and the determination of several types of biomolecules with high sensitivity. These techniques combine the low sample consumption and high separation power of CE with the selectivity of the probe to the target molecule. The assays can be defined according to the type of probe used: CE immunoassays, with an antibody as the probe, or aptamer-based CE, with an aptamer as the probe.

Online Enantioselective Capillary Electrophoretic Method for Screening Cytochrome P450 3A4 Inhibitors.

The market share of single-enantiomer drugs is steadily increasing. The pharmacodynamics and pharmacokinetics of individual enantiomers can differ considerably. Thus, their characteristics have to be addressed as early as possible in the development process of new pharmaceuticals.

Screening of Beta-Secretase Inhibitors by Capillary Electrophoresis-Mass Spectrometry.

Alzheimer's disease is the most common cause of dementia, currently afflicting almost 40 million patients worldwide. According to the amyloid cascade hypothesis, the pathogenesis of the disease could be slowed down or even stopped by the inhibition of beta-secretase, making this aspartic acid protease a potentially important drug target site. Capillary electrophoresis is a promising technique for screening putative enzyme inhibitors due to highly effective separations, minuscule sample and other chemicals consumption, compatibility with a variety of detection techniques, and high throughput via automation.

A novel method for classification of wine based on organic acids.

Bio-electronic tongue was linked to artificial intelligence processing unit and used for classification of wines based on carboxylic acids levels, which were indirectly related to malolactic fermentation. The system employed amperometric biosensors with lactate oxidase, sarcosine oxidase, and fumarase/sarcosine oxidase in the three sensing channels. The results were processed using two statistical methods - principal component analysis (PCA) and self-organized maps (SOM) in order to classify 31 wine samples from the South Moravia region in the Czech Republic.

Capillary electrophoresis integrated immobilized enzyme reactor for kinetic and inhibition assays of β-secretase as the Alzheimer's disease drug target.

Capillary electrophoresis integrated immobilized enzyme reactors are becoming an increasingly popular alternative for enzyme kinetic and inhibition assays thanks to their unique set of features including cost effectiveness, repeated use of the enzyme, minuscule sample consumption, rapid analysis time and easy automation. In this work we present the development and application of a capillary electrophoresis integrated immobilized enzyme reactor based on magnetic particles for kinetic and inhibition studies of β-secretase, a key enzyme in the development of Alzheimer's disease and a promising drug target. We document the optimization of the immobilization procedure, characterization of immobilized β-secretase, optimization of a mutually compatible incubation protocol and separation method as well as the production of the capillary electrophoresis integrated immobilized enzyme reactor.

Capillary electrophoresis-based approaches for the study of affinity interactions combined with various sensitive and nontraditional detection techniques.

Nearly all processes in living organisms are controlled and regulated by the synergy of many biomolecule interactions involving proteins, peptides, nucleic acids, nucleotides, saccharides, and small molecular weight ligands. There is growing interest in understanding them, not only for the purposes of interactomics as an essential part of system biology, but also in their further elucidation in disease pathology, diagnostics, and treatment. The necessity of detailed investigation of these interactions leads to the requirement of laboratory methods characterized by high efficiency and sensitivity.

A label-free MALDI TOF MS-based method for studying the kinetics and inhibitor screening of the Alzheimer's disease drug target β-secretase.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) is a well-established method with a unique set of qualities including sensitivity, minute sample consumption, and label-free detection, all of which are highly desired in enzyme assays. On the other hand, the application of MALDI TOF MS is usually limited by high concentrations of MS-incompatible compounds in the reaction mixture such as salts or organic solvents. Here, we introduce kinetic and inhibition studies of β-secretase (BACE1), a key enzyme of the progression of Alzheimer's disease.

Study of metabolic activity of human embryos focused on amino acids by capillary electrophoresis with light-emitting diode-induced fluorescence detection.

Assisted reproduction is a quickly developing field of reproductive medicine whose importance is growing every year due to the increasing number of patients suffering from infertility. As a result, there is a need for the continuous development and/or improvement of assisted reproductive technologies. This paper presents a new method for the in vitro measurement of the amino acid turnover of developing embryos based on capillary electrophoresis with light-emitting diode-induced fluorescence detection.

An improved design to capture magnetic microparticles for capillary electrophoresis based immobilized microenzyme reactors.

In this paper, we demonstrate the effectiveness of a new 3D printed magnet holder that enables capture of magnetic microparticles in commercially available capillary electrophoresis equipment with a liquid or air based coolant system. The design as well as the method to capture magnetic microparticles inside the capillary are discussed. This setup was tested at temperature and pH values suitable for performing enzymatic reactions.

In-depth insight into the methods of plasma protein-drug interaction studies: Comparison of capillary electrophoresis-frontal analysis, isothermal titration calorimetry, circular dichroism and equilibrium dialysis.

Plasma protein-drug binding assays are routinely performed during the early stages of drug discovery and development, which creates demand for an automated high-throughput screening assay to increase laboratory efficiency. A comprehensive comparison of the four methods typically used for determining the binding parameters is presented in this study with respect to the above demand. Capillary electrophoresis-frontal analysis, isothermal titration calorimetry, circular dichroism and equilibrium dialysis were used to study the affinity of human serum albumin for diclofenac and lidocaine.

Immobilized-enzyme reactors integrated with capillary electrophoresis for pharmaceutical research.

Enzymes play an essential role in many aspects of pharmaceutical research as drug targets, drug metabolizers, enzyme drugs and more. In this specific field, enzyme assays are required to meet a number of specific requirements, such as low cost, easy automation, and high reliability. The integration of an immobilized-enzyme reactor to capillary electrophoresis represents a unique approach to fulfilling these criteria by combining the benefits of enzyme immobilization, that is, increased stability and repeated use, as well as the minute sample consumption, short analysis time, and efficient analysis provided by capillary electrophoresis.