Figure 3. Effect of SPION concentration and washing on the activation of kallikrein.

A, Effect of washing on kallikrein activity of SPIONs. Nanoparticles were incubated with plasma or PBS and washed as described in Methods section. In parallel, plasma without added nanoparticles was processed in exactly the same way. Ten microliters of SPIONs or control plasma were incubated with 50 μl of the chromogenic substrate. The substrate conversion was monitored by reading the absorbance in the kinetic mode over 10 min period. All the samples were run in duplicates. Plasma-coated SPIONs (green triangles and black circles show different replicates) showed very strong enzymatic activity, while control plasma replicate (blue trace) and control SPIONs (red trace) show no enzymatic activity (only one replicate of each sample is shown due to the overlapping curves); B, SPIONs nanoparticles were incubated with plasma at different concentrations, washed 3 times by ultracentrifugation, and the kallikrein activity was visualized by the intensity of yellow color of the chromogenic substrate as described in Methods.