Quantification of azides on the surface of nanoparticles: towards precise bioconjugation

The precise design of bioconjugated nanoparticles is crucial for effective cell targeting and cellular uptake. Therefore, an accurate approach to creating and quantifying the organic ligand shell with a specific number of conjugated targeting ligands is essential. Click-chemistry has emerged as a robust method for bioconjugation, with azide-alkyne cycloaddition as the most prevalent method. Although nanoparticles are typically functionalized with azides, their quantification has rarely been reported. Here, we present two spectroscopic methods for the surface azide quantification of catechol-stabilized hafnium oxide nanoparticles as a model system. The first method exploits the intrinsic ability of catechol ligands to quench the fluorescence of conjugated fluorophores, whereas the second method represents a general strategy based on monitoring the alkyne absorbance during the click reaction. The latter method is independent of both inorganic core and the ligand shell, allowing it to be generally used. We also demonstrate the broader applicability on hafnium oxide nanoparticles capped with polyphosphonate ligands.