In-depth analysis of active compounds targeting tropomyosin-related kinase A via constructed lipid raft @capillary monolith affinity chromatography

In order to enrich the selection of biological ligands, realize the miniaturization analysis, and broaden the application of monolith materials for active ingredients screening and separating, we sough to construct a lipid raft @capillary monolith microcolumn affinity chromatography model. Single factor experiments and various characterization methods, including scanning electron microscopy (SEM) and thermogravimetric analysis, were employed to investigate the polymerization of the monolith column under different material ratios to determine optimal preparation conditions. Subsequently, the lipid raft from U251 cells was integrated with the monolith materials based on epoxy-based covalent crosslinking principle and characterized through SEM and immunofluorescence methods. Afterwards, the retention of positive drug gefitinib, negative drug gemcitabine and four licorice standards solution on the prepared lipid raft monolith microcolumn was then detected via electrochemical detection. The results exhibited that there was no specific adsorption for any active compounds on the blank monolith materials. Significantly, the lipid raft monolith microcolumn packed with TrkA-target proteins could be successfully validated for positive drug gefitinib with a high affinity sorption efficiency of 51.2%. This work expands the range of the utilization of affinity chromatography carriers and the selection of biological ligands, providing a new idea for the screening of active ingredients.