Analysis of VEGF–VEGFR2 Binding Kinetics Using Surface Plasmon Resonance

A gold sensor chip (GE Healthcare, USA) was modified to present an 11-mercaptoundecanoic acid (MUA, Aldrich) monolayer by injecting MUA solution into the flow cell in a Biacore 3000 (GE Healthcare, USA) for 30 min at 25 °C. The carboxylic groups of the MUA monolayer were then activated by flowing 0.2 M EDC and 0.05 M N-hydroxysuccinimide (NHS, Aldrich) solutions through the flow cell for 7 min. After activation, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE, Sigma) was chemically linked to the MUA layer by flowing DPPE solution until the response unit was saturated. The remaining NHS-ester groups on the MUA surface were blocked by injecting 1.0 M ethanolamine hydrochloride into the flow cell. Then the top layer was built by injecting a solution of recombinant human VEGFR-2 for 5 min. Finally, the mixture of VEGF and alginate sulfates or unmodified alginate suspended in PBS at a concentration of 2 μg/mL were injected into the flow cell to examine the association and dissociation rates of the VEGF complex with the gold sensor chip modified with DPPC and VEGFR-2. The media flow rate was kept constant at 5.0 μL/min. The kinetic data from SPR sensorgrams were obtained with the assistance of BIA evaluation version 4.1, where a 1:1 Langmuir binding model was applied to quantify the association and dissociation rates.²³