The precise design of bioconjugated nanoparticles is crucial for cell targeting and cellular uptake.[1] Therefore, an accurate approach to creating and quantifying the organic ligand shell with an ideal number of bioconjugated targeting ligands for efficient and cost-effective bioapplication is needed. In this respect, click-chemistry has emerged as one of the most reliable options for bioconjugation.[2] However, a precise determination of the number of reactive functional groups, typically azides, available for bioconjugation on the surface of NPs has been rarely reported. 

In this work, we synthesized catechol-stabilized hafnium oxide nanoparticles with different amount of azide functional groups (0-10 N₃/NP) on the surface as a model system to use in azide-alkyne cycloaddition reactions. We demonstrated that the NMR quantification of azide-carrying ligands, either in the bound state or stripped from the NP surface, comes with several unsolved challenges. To overcome them, we developed two convenient alternatives based on fluorescence or UV-Vis spectroscopy, both having similar accuracy and precision. The first quantification method is based on fluorescence quenching of a conjugated fluorophore by photoinduced charge transfer to nitrocatechol ligands. The second method is more general since it is based on the disappearance of the UV-Vis absorption band of the alkyne. Furthermore, we demonstrate the broader applicability of the second method on hafnium oxide nanocrystals capped with polyphosphonate ligands.

We believe that this quantitative analysis of surface functional groups will play an important role in precise bioconjugation, which is crucial for biomedical applications of NPs.

Figure 1. a) Schematic process behind the quantification method 1: DBCO-functionalized dye emits green light in the free form, but the fluorescence is quenched upon click reaction with surface azide groups due to PCT to acceptor nitrodopamine groups of the ligands; b) Chemical reaction behind the method 2: UV-tracking of azide-carrying nanoparticles with alkyne; c) Comparison of the quantification results by two developed methods.

[1] Makhani, E.Y., Zhang, A. & Haun, J.B. Nano Convergence 2021, 8, 38

[2] Thirumurugan P., Matosiuk D. and Jozwiak K. Chemical Reviews 2013, 113, 7, 4905–4979