Impact of Surface Functionalization on NV Quantum Properties: Implications for Biosensing with Fluorescent Nanodiamonds

Fluorescent nanodiamonds are rapidly redefining the frontiers of quantum sensing in biological systems, offering exceptional photostability, biocompatibility, and sensitivity through their nitrogen‐vacancy (NV) centers. However, unlocking their full potential as quantum sensors in living systems demands precise and robust covalent functionalization to ensure stable, targeted interaction with biomolecular structures without compromising NV performance. This work presents a systematic investigation of advanced surface modification strategies that can be utilized for quantum sensing in biological systems. Two bioorthogonal approaches are investigated: biotinylation, harnessing the ultra‐high affinity of the biotin‐streptavidin system, and maleimide conjugation, which enables site‐specific binding to cysteine‐tagged proteins. The influence of covalent surface modifications on the spin and optical properties of NV centers, specifically charge state stability and spin relaxation time, is examined to establish correlations between nanoscale chemical modification and quantum sensing performance. Photoluminescence spectroscopy, T 1 relaxometry, and advanced single‐particle measurements of fluorescence lifetimes using a confocal Anti‐Brownian Electrokinetic trap are employed to systematically investigate these properties in both individual nanodiamonds and particle ensembles. This work provides crucial insights into how the molecular architecture of the surface governs the charge stability and spin dynamics of NV centers, establishing a foundation for the rational engineering of diamond‐based quantum sensors.