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Magnetic microparticles as potential stationary phase for affinity studies in capillary electrochromatography

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Název česky Magnetické mikročástice jako potenciální stacionární fáze pro afinitní studie pomocí kapilární elektrochromatografie


Rok publikování 2023
Druh Další prezentace na konferencích
Fakulta / Pracoviště MU

Přírodovědecká fakulta

Popis The instrumentation of capillary electrophoresis (CE) enables introduction of novel materials as stationary phases into the capillary. The presence of selective stationary phase allows separation of analytes based on both, their mobility in electric field and interaction with stationary phase. Due to the combination of electrophoretic and chromatographic principles, this approach is called capillary electrochromatography (CEC). Columns used in CEC are usually prepared directly inside the capillary; the stationary phase is filling its interior or is bound on its inner surface. Superparamagnetic iron oxide particles represent novel stationary phase material with useful properties, besides their applicability for immobilized enzyme reactor formation. Magnetic behavior of the particles makes it easy to separate them from the reaction mixture during stationary phase preparation on their surface and enables formation of the column inside the capillary held by permanent magnets. Moreover, the column can be easily regenerated by the magnet removal. Convenient affinity modes of CE allow protein-ligand interactions studies in conditions close to physiological and with minimal sample consumption. Immobilization of protein on the surface of magnetic particles and preparation of affinity CEC column could expand the application potential of these methods. Protein of high interest in protein-ligand interaction studies is serum albumin. Since to its ability to bind drug molecules and its high plasma concentration, serum albumin affects significantly pharmacokinetical and pharmacodynamical profiles of drugs. According to the generally accepted “free drug” theory, only the free fraction of the drug is available for physiological processes resulting in its efficacy, and therefore it is important to understand interactions from all points of view. Successful covalent immobilization of bovine serum albumin (BSA) on the surface of magnetic silica coated particles has been demonstrated by our group in previous studies. The immobilized protein retained its drug binding properties. In the current study, magnetic particles with immobilized serum albumin were used for the preparation of an affinity CEC column, held inside the capillary by permanent magnets. Crucial and most tricky step was formation of stable and permeable column, as well as choice of suitable analysis conditions. Several magnet arrangements as external magnetic field source were tested since the particles tend to organize themselves into structures depending on magnetic field orientation. Prepared column was evaluated for its potential application in affinity studies and its suitability was evaluated with model drugs.
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