Influence of immobilized biomolecules on magnetic bead plug formation and retention in capillary electrophoresis

Abstract

Significant changes in the formation and retention of magnetic bead plugs in a capillary during electrophoresis were studied, and it was demonstrated that these effects were due to the type of biological molecule immobilized on the surface of these beads. Three biological molecules, an antibody, an oligonucleotide, and alkaline phosphatase (AP), were attached to otherwise identical streptavidin-coated magnetic beads through biotin-avidin binding in order to isolate differences in bead immobilization in a magnetic field resulting from the type of biological molecule immobilized on the bead surface. AP was also attached to the magnetic beads using epoxy groups on the bead surfaces (instead of avidin-biotin binding) to study the impact of immobilization chemistry. The formation and retention of magnetic bead plugs were studied quantitatively using light scattering detection of magnetic particles eluting from the bead plugs and qualitatively using microscopy. Both the types of biomolecule immobilized on the magnetic bead surface and the chemistry used to link the biomolecule to the magnetic bead impacted the formation and retention of the bead plugs.

Fig. 1

Diagram of magnet and capillary placement. Two NdFeB magnets with a surface field strength of 6403 Gauss were abutted to the capillary (360 μm od) at a 20° angle. This arrangement is based on the work of Slovakova et al. [20]. The dotted circle indicates the approximate field of view of the microscope. The microscope was located above (perpendicular to) the plane represented in the diagram.

Fig. 2

Electropherogram of streptavidin-coated magnetic beads with a biotinylated oligonucleotide bound to them (SA-Oligo). The neutral marker, coumarin, and beads were injected electrokinetically for 3.0 s and 10.0 s at 20.0 kV (351 V/cm), respectively. The voltage was then immediately reduced to 5.0 kV (88 V/cm) for transport to the magnetic immobilization area over 18 min. The applied potential was increased in steps, as indicated by the labeled, step-shaped plot above (electrophoretic current). The neutral maker elutes at 8.5 min and was detected by fluorescence. The beads eluted after increases in applied potential and were detected downstream of the magnetic immobilization area by light scattering.

Fig. 3

Images of initial bead plugs formed by a 10.0 s, 351 V/cm injection of 2.8 μm diameter beads, transported by 88 V/cm through a 50 μm id capillary and held in place by two NdFeB magnets as indicated in Fig. 1. The images were collected during electrophoresis at 88 V/cm. SA-Streptavidin, E-Epoxy, Oligo-Oligonucleotide, IgG-antibody, AP-Alkaline phosphatase.

Fig. 4

Magnetic bead elution with increasing electrophoretic potential for identical beads with different biological molecules immobilized on their surface. Beads were injected for 10.0 s at 351 V/cm and transported to the magnetic capture area by an applied potential of 88 V/cm. The applied potential was increased in steps of 88 V/cm (5.0 kV). Error bars are the standard deviation of n=3 experiments. Bead type abbreviations are defined in Fig. 3.

Fig. 5

Final bead plugs at 8.0 min during application of 439 V/cm. Bead type abbreviations are defined in Fig. 3.

Fig. 6

Comparison of the effect of immobilization chemistry on bead plug retention. Beads have alkaline phosphatase immobilized to either streptavidin (SA-AP) or epoxy (E-AP) surfaces. Beads were injected for 10.0 s at 351 V/cm and transported to the magnetic capture area by an applied potential of 88 V/cm. The applied potential was increased in steps of 88 V/cm (5.0 kV). Error bars are the standard deviation of n=3 experiments.