However, 100% coverage of the surface with functional ligands for click reactions could add additional costs to the production of the NPs, and also influences the colloidal stability of the NPs. For example, it was shown that the fraction of azide-carrying ligands must be kept below 15% to ensure good dispersibility in water over a long time. Low reactivity of surface groups due to steric hindrance comes as another limitation. For instance, gold nanoparticles could only accommodate 8 molecules of fluorescent dye despite having 117 azide groups on the surface. Of the few reports that do quantify the azide ligand density, the azide quantification is typically based on photo-or fluorometric detection of a dye label. Particles with a certain number of azide groups are left to react with the DBCO derivative of a fluorescent dye, followed by the purification of the labeled nanoparticles and separate analysis of the nanoparticles and/or supernatant to determine the amount of bound or unbound dye. More sophisticated variations of this method include the use of cleavable reagents that are first clicked to the surface. After purification, the dye is released into the solvent by cleaving another weak point in the molecule (e.g., hydrolysis of an amide). The obvious disadvantage of relying on dye labels is the need for extensive purification of the sample to ensure the removal of all unreacted labels, as there is no optical difference between bound and free dye. The Förster Resonance Energy Transfer (FRET) effect can be used to circumvent this issue, but it is limited to the quantification of optically active nanoparticles, such as quantum dots. In addition, direct quantification is only possible when the light scattering by NPs is negligible and dye-dye interactions can be excluded because of the low functional group density on the surface. Another option is quantitative nuclear magnetic resonance (qNMR), which is typically used to analyze the organic ligand shell of nanoparticles and to determine the corresponding ligand densities. For example, this method was used for the analysis of gold nanoparticles stabilized with an azide-carrying peptide. However, as the NMR signals get significantly broadened when the ligands are bound to the nanoparticle surface, the analysis becomes complicated in the case of mixed ligand shells, especially, in the case of a low ratio of azidecarrying ligands to stabilizing ligands. qNMR can also be performed on ligands released to the solution following the addition of stronger ligands or decomposition of the NPs, but the complete removal and recovery into solution of the ligands can be problematic, especially in the case of strongly binding ligands. The limitations of NMR also include the relatively high amount of material needed for analysis compared to optical methods, as well as the inability to study NPs with paramagnetic properties, such as iron oxide, widely used in MRI imaging, or lanthanide-doped NPs.