2.4. Ninhydrin Colorimetric Assay

The concentration of the free amine groups on the MNPs’ surface was determined by a ninhydrin colorimetric assay [39]. The method is based on the formation of Ruhemann’s purple complex, which is indicative of the reaction between amino groups and ninhydrin. The presence of the complex can be detected by UV–Vis at 600 nm. For the calibration curve, aliquots of ODA (0.1–0.6 mL, 0.25 mg mL⁻¹) in dimethylformamide (DMF) were inserted into a series of tubes. To each tube, ninhydrin solution in MeOH (0.06 m, 0.7 mL, 10.7 mg mL⁻¹) was added, and the mixture was mixed well and heated at 100 °C for 5 min. After heating, the tubes were left to cool, and the contents were transferred to a 5 mL volumetric flask and diluted with DMF (2 mL), and then the UV–Vis absorbance was measured. A solution of particles in DMF (0.25 mg mL⁻¹) was prepared accordingly, and 0.4 mL of the sample stock solution was transferred into boiling tubes with a ninhydrin solution (0.7 mL) by following the same procedure described above, and UV–Vis measurements of the formed complex were performed. DMF was chosen because it leads to good dispersions of particles.