2.2.5. Immobilization of Capture Antibody to Beads

Dynabeads (M-280) streptavidin-coated magnetic microbeads were conjugated with biotinylated anti-HIV-1 gp120 antibody for visualization of the captured HIV-1 virions inside a microchip. Figure S1 demonstrates the process of antibody conjugation to microbeads. Dynabeads are critical for this application due to their high refractive index. The refractive index is an intrinsic material property of microbeads that provides invaluable information for various imaging and biosensing applications [36]. Dynabeads have a higher refractive index than silica microbeads as per the company’s provided information. As a result of the higher value of the refractive index, Dynabeads have a characteristic advantage of better detection.

In order to attach the antibody to these beads, we have utilized the manufacturer protocol. Initially, the stock solution containing the beads was vortexed for 30 s. 100 μL of the microbeads were collected in an Eppendorf Protein LoBind tube (14-282-304, Fisher Scientific). One mL of washing buffer (PBS with pH7.4) was added to the microbeads. The vial containing the beads and washing buffer was placed in the close vicinity of a permanent external magnet for one minute. The microbeads were attracted to the permanent magnet and they formed a pellet. The supernatant was removed with the help of a pipette. The microbeads were resuspended in the 100 microliters washing buffer. This washing process was repeated twice. As per the manufacturer’s specifications, 10 μg of biotinylated anti-HIV1 gp120 antibody was added to these washed microbeads. The sample was incubated on a shaker (15 RPM) for 30 min at room temperature. As a result of the very strong interaction between biotin and streptavidin, antibody coating of the Dyna beads was accomplished. The antibody immobilized magnetic beads were collected with the help of an external permanent magnet, and the supernatant was discarded. The antibody-conjugated magnetic microbeads were washed three times. The unoccupied sites of these antibody-coated beads were blocked with 4% BSA solution. This blocking was done overnight at 4 °C on a shaker. The microbeads were collected with the help of a permanent magnet and washed again with PBS. The blocked antibody-coated magnetic microbeads were resuspended in PBS solution and stored at 4 °C for further downstream applications.