Online concentration and affinity separation of biomolecules using multifunctional particles in capillary electrophoresis under magnetic field

To overcome several problems in affinity capillary electrophoresis (ACE), i.e., low detectability, need for sample derivatization, and difficulty in the fixation of affinity ligands (ALs), multifunctional magnetic particles (MFMPs) were prepared by immobilizing both fluorescent molecules and ALs for low-density lipoproteins onto the surface of magnetic polymer microspheres with a polyelectrolyte multilayer coating technique and applied to the ACE analysis. The prepared MFMPs showed a remarkable change in the electrophoretic mobility (mu(ep)) by the addition of low-density lipoproteins (LDL), whereas for high-density lipoproteins (HDL), mu(ep) of the MFMPs kept constant, so that it was confirmed that the MFMPs possess an affinity with LDL. On the other hand, the MFMPs can be trapped by the magnetic field even under a higher electric field for electrophoresis. By a successive on-off control of the magnetic field, online preconcentration of the LDL bound MFMPs and the selective separation of LDL from HDL were successfully achieved. In the ACE analysis of LDL employing UV detection, an 82-fold increase in the sensitivity was obtained by the on-capillary sample preconcentration using the MFMPs. When laser induced-fluorescence detection was employed, furthermore, the limit of detection for LDL was improved to the order of subpicomolar.