Linker‐Free Covalent DNA Functionalization of Quantum‐Grade Nanodiamonds
At a Glance
Section titled “At a Glance”| Metadata | Details |
|---|---|
| Publication Date | 2025-10-24 |
| Journal | Advanced Optical Materials |
| Authors | Jakub Čopák, Frederik Steiner, Ema Fialova, Tomás̆ Matous̆ek, Jan Plutnar |
| Institutions | Czech Academy of Sciences, Institute of Organic Chemistry and Biochemistry, Charles University |
| Analysis | Full AI Review Included |
Executive Summary
Section titled “Executive Summary”This research establishes a robust, linker-free covalent functionalization strategy for quantum-grade nanodiamonds (NDs) to create highly sensitive quantum biosensors.
- Core Achievement: Successful direct covalent attachment of DNA oligonucleotides to the sp3 carbon lattice of NDs, bypassing conventional linkers that compromise NV center sensitivity.
- Optimal Methodology: Radical Decarboxylative Azidation (AZIDDEC) of tri-acid oxidized NDs, followed by strain-promoted azide-alkyne cycloaddition (SPAAC) using DBCO-modified DNA.
- High Conjugation Yield: The optimized protocol achieved a high-density loading of approximately ≈640 DNA strands per ND particle.
- Quantum Property Preservation: The NV negative charge state (NV-) stability was preserved and slightly improved (NV-/NV0 ratio increased to 1.31) after DNA clicking.
- Enhanced Relaxation Time: The spin-lattice relaxation time (T1), critical for quantum sensing, was restored and extended to 264 µs after conjugation, confirming the preservation of quantum performance.
- Stability: The resulting DNA-conjugated NDs demonstrated excellent colloidal and chemical stability in aqueous solution for over 22 months.
Technical Specifications
Section titled “Technical Specifications”| Parameter | Value | Unit | Context |
|---|---|---|---|
| ND Precursor Material | HPHT NDs (MSY 0 - 0.05) | N.A. | Microdiamant |
| NV Center Creation | 16.5 MeV, 4 C cm-2 | N.A. | Electron beam irradiation |
| Optimal T1 Relaxation Time | 264 ± 54 | µs | AZIDDEC-Click (DNA conjugated) |
| Precursor T1 Relaxation Time | 218 ± 35 | µs | 3ACID (Tri-acid oxidized) |
| Maximum DNA Loading | ≈640 | strands/ND | Achieved via heating in DMSO |
| Azide FTIR Signature | 2133 | cm-1 | Asymmetric stretch of the azide group |
| Hydrodynamic Diameter (DH) | 74.9 ± 2.0 | nm | AZIDDEC-Click sample |
| Zeta Potential (ζ) | -32.6 ± 0.6 | mV | AZIDDEC-Click sample |
| NV-/NV0 PL Ratio (Max) | 1.31 ± 0.06 | N.A. | AZIDDEC-Click sample (Improved stability) |
| Nitrogen Content (AZIDDEC) | 0.22 ± 0.06 | wt.% | Decarboxylative route |
| Oxidation Temperature (Tri-Acid) | 90 | °C | Used to maximize surface carboxyls |
Key Methodologies
Section titled “Key Methodologies”The study compared two routes for azide functionalization: nucleophilic substitution (targeting hydroxyls) and radical decarboxylative azidation (targeting carboxyls). The latter proved superior for reactivity.
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ND Precursor Preparation (3ACID):
- HPHT NDs were air-oxidized (540 °C, 6 h) to remove sp2 carbon.
- NDs were then tri-acid oxidized using H2SO4, HClO4, and HNO3 at 90 °C for 48 h to generate a high density of surface carboxyl groups.
- NV centers were created via electron irradiation (16.5 MeV) followed by 900 °C annealing.
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Optimal Azidation (Radical Decarboxylative Azidation):
- The carboxyl-rich 3ACID NDs were suspended in a 50% acetonitrile/water solution.
- The reaction utilized tosyl azide (TsN3) as the azide source, catalyzed by silver (I) fluoride (AgF) and initiated by potassium persulfate (K2S2O8).
- Conditions: Stirred at 50 °C for 24 h under an argon atmosphere.
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Bioconjugation (SPAAC Click Reaction):
- Azide-terminated NDs (AZIDDEC) were reacted with dibenzocyclooctyne (DBCO)-modified oligonucleotides (DBCO-PEG4-DNA-Gd).
- High-Yield Protocol: Incubation in dimethyl sulfoxide (DMSO) at 37 °C for 7 days, achieving ≈640 strands/ND.
- Fast Protocol: Freeze-thaw cycling in DMSO/water mixture, achieving ≈400 strands/ND in 24 h.
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Quantum Characterization:
- Single-particle photoluminescence (PL) spectra were measured using a 532 nm laser (40 µW power).
- NV-/NV0 ratios were determined by spectral deconvolution using nonnegative matrix factorization.
- T1 relaxometry was performed using an all-optical sequence with low laser power to mitigate laser-induced charge effects.
Commercial Applications
Section titled “Commercial Applications”The developed linker-free functionalization technology is critical for advancing diamond-based quantum technologies in biological and chemical sensing.
- Quantum Biosensing: Enables the creation of highly selective, high-sensitivity quantum probes for real-time monitoring of spin interactions in biological environments, leveraging the maximal NV-to-analyte proximity.
- In Vivo/Intracellular Sensing: The stability, nanoscale size, and simple all-optical T1 readout sequence make these NDs ideal for high spatial and temporal resolution measurements inside living cells.
- Targeted Diagnostics: Used to construct sequence-selective DNA or aptamer arrays directly on the ND surface, facilitating targeted detection of specific nucleic acids or proteins.
- Biomolecule Conjugation: The bioorthogonal click chemistry approach provides a universal platform for robust, covalent attachment of various macromolecules (e.g., proteins, peptides, polymers) to sp3 diamond surfaces for drug delivery or advanced materials science.
- Long-Term Storage and Use: The demonstrated stability of the conjugates (stable for >22 months) supports commercial applications requiring long shelf life and reliable performance in aqueous media.
View Original Abstract
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.