Functionalization of Magnetic Nanoparticles

Fifty microliter of commercial 250 nm Ni–NTA-coated MNPs (Nanomag‐D, Micromod, 4.9 × 10¹¹ particles mL⁻¹) were washed twice with 500 µL of 0.1 M PB Tween 20 (Scharlab) (0.05%, v v⁻¹) by removing the supernatant after trapping the particles with a magnetic concentrator (Dynal‐Biotech). Then, 500 µL of the recombinant RBP Gp17, expressed and purified as described by Costa et al. [20 (link)], at a final concentration of 5 µM was added to the MNPs and incubated in an orbital shaker at 500 rpm, 20 °C for 2 h. After MNP functionalization, the above-mentioned washing procedure was performed to remove the supernatant containing the unbound protein. In the next step, 500 µL of a 5% (w v⁻¹) bovine serum albumin (BSA) (Sigma-Aldrich) solution in 0.1 M PB was added to the MNPs to block the remaining free surface and incubated at the same conditions as during functionalization for 1 h. The supernatant was discarded and the MNP-RBPs conjugates were washed with 0.1 M PB Tween 20 (Scharlab) (0.05% v v⁻¹), resuspended in 50 µL 0.1 M PB, and kept at 4 °C as stock. For comparison purposes, 50 µL of commercial 250 nm streptavidin-coated MNPs (Nanomag‐D, Micromod, 4.9 × 10¹¹ MNPS mL⁻¹) were functionalized with 500 µL of a biotinylated E. coli polyclonal antibody (LS-C56164, LSBio) at a final concentration of 50 μg mL⁻¹, following the same procedure described above.