Linker‐Free Covalent DNA Functionalization of Quantum‐Grade Nanodiamonds

Abstract Nanodiamonds (NDs) with nitrogen‐vacancy (NV) centers are promising quantum sensors for biological environments, utilizing the NV spin relaxation susceptibility to its environment. Since unfunctionalized ND probes cannot directly discriminate among the many contributors to the relaxation signal, surface functionalization is essential for molecular recognition. Conventional modification strategies often introduce sp ² carbon or rely on linkers, both of which compromise sensitivity. Direct azidation of the sp ³ lattice is therefore pursued to support click‐based covalent coupling. Two complementary routes are explored: i) nucleophilic substitution of a brominated surface and ii) radical decarboxylative azidation. Both methods yield azide‐terminated NDs without detectable lattice degradation. Surface reactivity is assessed through model click reactions with Alexa Fluor 488‐alkyne and a dibenzocyclooctyne‐modified oligonucleotide. Various reaction settings are evaluated under multiple conditions (freeze‐thaw or heating, water or dimethylsulfoxide as solvent). The decarboxylative route, followed by alkyne coupling in dimethylsulfoxide at elevated temperature, afforded the highest conjugation efficiency. Crucially, the NV negative charge state and spin‐lattice relaxation time ( T ₁ ) are preserved after azidation and slightly improved after clicking. Comprehensive characterization confirms these findings. Optimized protocols covalently attached ≈640 DNA strands per ND. In summary, the linker‐free, bioorthogonal strategy supplies robust functionalization with biomolecules for next‐generation NV‐based quantum biosensors.