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. As a result, many instrumental approaches differing in their fundamental principles have been developed, including those based on capillary electrophoresis. Although capillary electrophoresis offers numerous advantages for such studies, it still has one serious limitation, its poor concentration sensitivity with the most commonly used detection method–ultraviolet‐visible spectrometry. However, coupling capillary electrophoresis with a more sensitive detector fulfils the above‐mentioned requirement. In this review, capillary electrophoresis combined with fluorescence, mass spectrometry, and several nontraditional detection techniques in affinity interaction studies are summarized and discussed, together with the possibility of conducting these measurements in microchip format.

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. Besides its efficacy, these interactions subsequently influence the liberation, absorption, distribution, metabolism, excretion, and toxicological properties of the drug. It is important to not only determine the binding strength and stoichiometry, but also the binding site of a drug on the plasma protein molecule, because the co-administration of drugs with the same binding site can affect the above-mentioned concentration and as a result the pharmacological behavior of the drugs and lead to side effects caused by the change in free drug concentration, its toxicity. In this study, the binding characteristics of six drugs with human serum albumin, the most abundant protein in human plasma, were determined by capillary electrophoresis-frontal analysis, and the obtained values of binding parameters were compared with the literature data. The effect of several drugs and site markers on the binding of l-tryptophan and lidocaine to human serum albumin was investigated in subsequent displacement studies which thus demonstrated the usability of capillary electrophoresis as an automated high-throughput screening method for drug-protein binding studies.

Capillary electrophoresis-based immunoassay and aptamer assay: A reviewOver 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. Immunoassays are generally divided into homogeneous and heterogeneous groups, and homogeneous variant can be further performed in competitive or noncompetitive formats. Interacting partners are free in solution at homogeneous assay, as opposed to heterogeneous analyses, where one of them is immobilized onto a solid support. Highly sensitive fluorescence, chemiluminescence or electrochemical detections were typically used in this type of study. The use of the aptamers as probes has several advantages over antibodies such as shorter generation time, higher thermal stability, lower price, and lower variability. The aptamer-based CE technique was in practice utilized for the determination of proteins in biological fluids and environmentally or clinically important small molecules. Both techniques were also transferred to microchip. This review is focused on theoretical principles of these techniques and a summary of their applications in research.Abbreviations: Ag, antigen; CEIA, CE immunoassay; MCE, microchip CE; SELEX, systematic evolution of ligands by exponential enrichment and they are produced by the immune system of living organisms. Ag that start the immunological response are also called immunogens. These Ab-Ag complexes are stabilized by dipole-dipole, van der Waals or hydrophobic interactions and hydrogen bonds [5,6].The most often used immunoassays are ELISA with Ab or Ag immobilized onto solid support (plates, glass fibers, or plastic tubes), immunofluorescence assay, fluorescence polarization immunoassay, luminescence immunoassay, microparticle enzyme immunoassay, and biosensors. The major disadvantages of immunoassays are the tedious process, time-consuming reactions, and problems with reproducibility and nonspecific interactions [6].CE is a powerful analytical method for the separation of a wide range of molecules. The coupling of CE and immunoassay in the new CE immunoassay (CEIA) technique provided both separation power and specificity of interactions. Other advantages are the possibility of multianalyte analyses, automation, low sample consumption, high speed, flexibility, a lower amount of false-positive results, and also the possibility of connection with highly sensitive detectors [5].In CEIA, the Fab fragments of Ab containing Ag-specific regions are more often used than the whole monoclonal and Color online: See article online to view Figs. 4 and 7 in color.

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. These model drugs were chosen due to their different physico-chemical properties and different binding sites on the albumin molecule, also resulting in different binding strength. The binding parameters estimated under the conditions as similar as possible were comparable among all these approaches as well as to the literature values. Besides this, the comparison of the results and especially other considerations demonstrated the benefits and drawbacks of the selected methods, with capillary electrophoresis-frontal analysis being the best candidate for such studies.

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