Linker‐Free Covalent DNA Functionalization of Quantum‐Grade Nanodiamonds

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

Technical Documentation & Analysis: Linker-Free Covalent Functionalization of Quantum-Grade Nanodiamonds

Executive Summary

This research validates a critical, linker-free surface functionalization strategy for Nanodiamonds (NDs) that is highly relevant for next-generation quantum biosensors. The key findings and value proposition for 6CCVD clients are summarized below:

Preserved Quantum Properties: The optimized method successfully preserves the NV negative charge state (NV^-) and significantly improves the spin-lattice relaxation time (T₁), achieving 264 ± 54 µs post-conjugation.
Linker-Free Covalent Coupling: The strategy avoids conventional linkers and sp² carbon introduction, ensuring the closest possible proximity between the NV center and the analyte, maximizing quantum sensing sensitivity (which follows an inverse sixth-power distance dependence).
High Conjugation Efficiency: Radical decarboxylative azidation (AZID_DEC) followed by SPAAC click chemistry achieved robust, high-yield attachment of biomolecules (up to $\approx$640 DNA strands per ND).
CVD Transferability: The authors explicitly confirm that this sp³-carbon surface chemistry is transferable from HPHT NDs to Chemical Vapor Deposition (CVD) diamond, directly enabling 6CCVD’s MPCVD materials.
Robust Biocompatibility: DNA-conjugated NDs demonstrated excellent colloidal and chemical stability in aqueous solution for over 22 months, confirming suitability for long-term biological applications.
Enabling Technology: This methodology is pivotal for constructing sequence-selective DNA arrays and highly specific NV-based quantum probes for molecular sensing.

Technical Specifications

The following hard data points were extracted, demonstrating the material properties and performance metrics achieved through the optimized functionalization route (AZID_DEC-Click).

Parameter	Value	Unit	Context
Initial T₁ (3ACID Precursor)	218 ± 35	µs	Tri-acid oxidized starting material
Optimized T₁ (AZID_DEC-Click)	264 ± 54	µs	Post-linker-free DNA conjugation
NV^-/NV⁰ PL Ratio (3ACID)	1.18 ± 0.10	Ratio	Starting material
NV^-/NV⁰ PL Ratio (AZID_DEC-Click)	1.31 ± 0.06	Ratio	Improved NV^- stability post-conjugation
Maximum Conjugation Yield	$\approx$640	Strands/ND	DBCO-PEG₄-COOH click, elevated temperature
Optimal Reaction Temperature (SPAAC)	37	°C	For biomolecule conjugation
Optimal Reaction Time (SPAAC)	7	Days	For maximum yield (640 strands/ND)
Initial Hydrodynamic Diameter (3ACID)	60.1 ± 0.6	nm	Tri-acid oxidized precursor
Final Hydrodynamic Diameter (AZID_DEC-Click)	74.9 ± 2.0	nm	DNA-conjugated NDs (Colloidally stable)
Final Zeta Potential (AZID_DEC-Click)	-32.6 ± 0.6	mV	Indicates preserved negative surface charge

Key Methodologies

The research systematically compared two azidation routes to achieve direct covalent coupling to the sp³ diamond lattice. The optimal route utilized radical decarboxylative azidation on highly oxidized surfaces.

Preparation of Carboxyl-Rich Precursor (3ACID):
- Air-oxidized NDs were subjected to aggressive tri-acid oxidation (HNO₃/HClO₄/H₂SO₄) at 90 °C for 48 h to maximize surface carboxylic acid groups (COOH).
Optimal Azidation (Radical Decarboxylative Route):
- The 3ACID sample was reacted with Tosyl Azide (TsN₃) in the presence of Silver Fluoride (AgF) and Potassium Persulfate (K₂S₂O₈) initiator.
- Recipe Parameters: 50 °C for 24 h under an argon atmosphere.
- Result: Yielded AZID_DEC, which showed lower nitrogen content (0.22 wt.%) than the nucleophilic route but significantly higher click reactivity due to better steric accessibility.
Bioconjugation via SPAAC Click Chemistry:
- The AZID_DEC NDs were conjugated with DBCO-PEG₄-DNA-Gd oligonucleotide.
- Optimal Conditions: Dimethylsulfoxide (DMSO) solvent, 37 °C incubation for 7 days.
- Result: Achieved the highest conjugation yield ($\approx$640 strands/ND) while maintaining the NV^- charge state and improving T₁.
Characterization Techniques:
- Fourier-transform infrared spectroscopy (FTIR) confirmed the azide stretch at 2133 cm^-1.
- X-ray photoelectron spectroscopy (XPS) confirmed the dominance of the sp³ carbon component post-functionalization.
- All-optical T₁ relaxometry confirmed preserved quantum coherence times at the single-particle level.

6CCVD Solutions & Capabilities

6CCVD provides the high-purity, quantum-grade MPCVD diamond materials necessary to replicate and scale this linker-free functionalization strategy for commercial quantum biosensor development.

Applicable Materials

The research confirms that the sp³-carbon surface chemistry is transferable to CVD diamond. 6CCVD specializes in the materials required for high-performance NV center applications:

Quantum Grade Single Crystal Diamond (SCD): For applications requiring the highest purity, lowest defect density, and maximum T₁ coherence times. 6CCVD offers SCD plates up to 500 µm thick, ideal for creating large-area quantum sensors.
Polycrystalline Diamond (PCD) Wafers: For large-scale, cost-effective sensor arrays. We offer PCD wafers up to 125mm in diameter, suitable for high-throughput surface modification and device integration.
Boron-Doped Diamond (BDD): While not the focus of this paper, BDD substrates are available for electrochemical sensing applications that may require similar surface functionalization techniques.

Customization Potential

6CCVD’s in-house engineering and fabrication capabilities directly support the advanced requirements of quantum sensing research:

Research Requirement	6CCVD Custom Capability	Benefit to Client
Surface Precursor Preparation	Custom Surface Termination	We provide precise oxygen-terminated surfaces (analogous to the 3ACID precursor) or hydrogen-terminated surfaces, ready for immediate chemical processing.
Device Integration	Custom Metalization Services	Internal capability for depositing Au, Pt, Pd, Ti, W, and Cu contacts, essential for integrating diamond sensors into microfluidic or electronic architectures.
Large-Area Scaling	Custom Dimensions & Thickness	Plates/wafers available up to 125mm (PCD) and SCD thicknesses from 0.1 µm to 500 µm, enabling scale-up from research to production.
Surface Quality	Ultra-Low Roughness Polishing	SCD polishing to Ra < 1nm and inch-size PCD polishing to Ra < 5nm, ensuring minimal surface defects that could compromise NV center stability or chemical uniformity.

Engineering Support

6CCVD’s in-house PhD team offers authoritative professional support to accelerate your research:

Material Selection for Quantum Projects: Assistance with selecting the optimal diamond type (SCD vs. PCD) and nitrogen concentration to maximize NV center density and T₁ performance for specific [Quantum Biosensor] projects.
Surface Chemistry Consultation: Guidance on optimizing surface preparation recipes (e.g., oxidation parameters) to maximize the density of reactive groups (like COOH) needed for high-yield decarboxylative azidation.
Integration Support: Expertise in integrating custom metal contacts and defining precise geometries via laser cutting for advanced device architectures utilizing this linker-free functionalization.

For custom specifications or material consultation, visit 6ccvd.com or contact our engineering team directly.

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.

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Original Source

DOI: https://doi.org/10.1002/adom.202502279