Biacore reagents

Surface plasmon resonance (SPR) experiments were conducted in a Biacore T200 instrument (GE Healthcare Life Sciences). During measurements, the flow buffer HBS-N was used (10 mM HEPES, pH 7.4 with 150 mM NaCl). Immobilization of bovine folate binding protein (FBP, Sigma-Aldrich) was carried out at 25°C by an EDC-based amide coupling method using standard Biacore reagents (GE Healthcare Life Sciences). FBP-presenting chips (CM5) were prepared with protein density at 9.03 FBP ng/mm². SPR experiments were performed by injection of a ligand solution, HES or HES-MTX nanoconjugate, each prepared in HBS-N buffer (concentrations were presented as MTX-equiv), at a flow rate of 40 µl/min. The conjugates were injected over a reference channel and over a channel with immobilized FBP for 200 sec. Each analysis cycle consisted of a 60 sec initial period, in which the stability of the baseline was monitored. The injection of buffer was performed between each analysis cycle for a double reference. At the end of each dissociation phase, the chip surface was treated with 10 µl of 10 mM glycine-HCl (pH 2.5) for surface regeneration. Sensorgrams for the reference channel were subtracted from sensorgrams for the channel with FBP. Subsequently, sensorgrams of buffer were subtracted from sensorgrams of the HES-MTX conjugates.