Electron Paramagnetic Resonance of a Single NV Nanodiamond Attached to an Individual Biomolecule

At a Glance

Metadata	Details
Publication Date	2016-05-01
Journal	Biophysical Journal
Authors	Richelle M. Teeling-Smith, Young Woo Jung, Nicolas Scozzaro, Jeremy Cardellino, Isaac V. Rampersaud
Institutions	The Ohio State University, Samsung (South Korea)
Citations	18
Analysis	Full AI Review Included

Technical Documentation & Analysis: Single Molecule EPR using NV Nanodiamonds

This document analyzes the research demonstrating single-molecule Electron Paramagnetic Resonance (smEPR) using Nitrogen-Vacancy (NV) nanodiamonds attached to DNA. It highlights the material requirements and positions 6CCVD’s high-purity MPCVD diamond solutions as the optimal path for replicating and advancing this quantum sensing methodology.

Executive Summary

This research establishes a foundational methodology for single-molecule magnetic resonance studies by leveraging the high sensitivity of NV centers in nanodiamonds.

Sensitivity Breakthrough: The study successfully performed Electron Paramagnetic Resonance (EPR) measurements on a single, labeled biomolecule (double-stranded DNA), overcoming the severe sensitivity limitations (10¹⁰-10¹⁵ molecules required) of conventional inductive EPR detection.
NV Probe Functionality: Single-crystal nanodiamonds (10-200 nm) containing NV centers served as optically detectable spin probes, site-specifically attached to DNA via biotin-streptavidin chemistry.
Detection Method: Continuous Wave Optically Detected Magnetic Resonance (ODMR) was used to measure the paramagnetic resonance of the NV spins.
Rotational Dynamics: The nanodiamond probe demonstrated isotropic rotation on timescales ($\tau_R \approx 100$ µsec) slower than the spin relaxation time ($T_2 \approx 0.25-1.4$ µs), resulting in a powder spectrum equivalent.
Future Requirement: Achieving motional narrowing (where $\tau_R \le T_2$) to measure faster biomolecular dynamics requires significantly smaller (down to 10 nm) and higher-purity nanodiamonds to maximize $T_2$ coherence times.
Application Potential: This methodology is broadly applicable for investigating the structural dynamics of complex biomolecular systems, including RNA, proteins, and lipid vesicles.

Technical Specifications

The following table summarizes the critical material and experimental parameters extracted from the research paper.

Parameter	Value	Unit	Context
Nanodiamond Synthesis Method	HPHT (Van Moppes SYP0.09)	N/A	Source material for nanodiamonds.
Nanodiamond Size Range	10 - 200	nm	Diameter of single-crystal nanodiamonds used.
Initial Nitrogen Impurity Content (n_N)	~200	ppm	Concentration in the bulk HPHT diamond.
Final NV Center Density	~30	ppm	Concentration after irradiation and annealing.
Electron Irradiation Energy	1.5	MeV	Used to create vacancies for NV formation.
Annealing Temperature	900	°C	Post-irradiation thermal treatment (3 hours).
NV Zero-Field Splitting (D)	2.87	GHz	Intrinsic splitting of the NV electronic ground state.
Excitation Wavelength	532	nm	Continuous wave DPSS laser for optical excitation.
Applied Magnetic Fields (B)	0, 18.7 ± 0.05, 32.6 ± 0.09	Gauss	Fields used for powder spectra fitting.
Transverse Spin Relaxation Time (T₂)	0.25 - 1.4	µs	Typical T₂ range for NV centers in nanodiamonds.
Target T₂ for Motional Narrowing	0.7	msec	Reported T₂ for highly pure nanodiamonds in aqueous environments.
Rotational Correlation Time ($\tau_R$)	~100	µsec	Estimated for the ~100 nm nanodiamond probe.

Key Methodologies

The experiment relied on precise material engineering, chemical functionalization, and integrated quantum sensing hardware.

NV Center Fabrication: High Pressure High Temperature (HPHT) diamond powder (200 ppm N) was micro-fractured (10-200 nm), irradiated with a 1.5 MeV electron beam (3.48x10¹⁸/cm² per hour), and annealed at 900 °C (96% Ar, 4% H₂) to achieve a target NV density of ~30 ppm.
Surface Preparation: Nanodiamonds underwent rigorous acid-reflux cleaning (H₂SO₄/HNO₃, NaOH, HCl at 90 °C) to remove graphitic residue (sp² carbon) and terminate the surface with carboxyl groups, crucial for subsequent functionalization and reducing fluorescence damping.
Site-Specific Biotinylation: The carboxyl-terminated surface was sequentially reacted with glycidol (to introduce hydroxyl groups), N’-disuccinimidyl carbonate (DSC), 4,7,10-Trioxa-1,13-tridecanediamine (TTDD), and NHS-dPEG¹²-biotin to covalently attach biotin, ensuring efficient streptavidin binding.
Single Molecule Tethering: Dual-labeled Lambda DNA (biotin/digoxigenin) was tethered between the streptavidin-coated NV nanodiamond and an anti-digoxigenin coated glass flow cell surface, creating a stable, single-molecule system.
ODMR Measurement: A custom confocal microscope integrated optical excitation (532 nm laser) and fluorescence collection with a coplanar waveguide microwave circuit (Au) to perform continuous wave ODMR measurements in a controlled aqueous buffer environment.

6CCVD Solutions & Capabilities

The research highlights the critical need for ultra-pure diamond material to maximize the spin coherence time ($T_2$) and enable the measurement of faster biomolecular dynamics ($\tau_R \le T_2$). 6CCVD’s expertise in high-purity MPCVD diamond growth directly addresses these limitations, providing the necessary foundation for next-generation single-molecule quantum sensing.

Research Requirement / Challenge	6CCVD Solution & Capability	Technical Advantage for Quantum Sensing
High Purity Material for Extended T₂ (Paper used 200 ppm N HPHT; needs < 1 ppm N for msec T₂)	Optical Grade Single Crystal Diamond (SCD)	6CCVD specializes in MPCVD growth with nitrogen impurity levels controlled to sub-ppb concentrations. This ultra-low background nitrogen is essential for minimizing spin bath decoherence, yielding the long $T_2$ times (up to 0.7 msec) required for motional narrowing and high-resolution pulsed EPR.
Custom NV Concentration & Depth (Paper used bulk irradiation/annealing)	Tailored SCD Substrates & Post-Processing	We provide SCD plates (up to 125mm) optimized for precise NV creation. We offer controlled nitrogen doping (if required) and can assist in developing specific irradiation/annealing recipes to achieve optimal NV density and shallow implantation depths for enhanced surface sensitivity.
Integrated Microwave Circuitry (Paper used Au coplanar waveguide)	In-House Custom Metalization Services	6CCVD offers internal metalization capabilities (Au, Pt, Ti, Pd, W, Cu). We can deposit high-quality metal films directly onto SCD substrates, enabling the fabrication of integrated coplanar waveguides and microwave structures for robust, high-power ODMR/EPR experiments.
Nanodiamond Precursor Material (Need for ultra-pure, size-controlled NDs)	High-Quality SCD Plates for Top-Down Fabrication	We supply high-purity, low-strain SCD wafers (up to 500 µm thick) ideal for subsequent top-down processing (e.g., reactive ion etching, milling) to create ultra-pure nanodiamonds with precise size control (e.g., the target 10 nm size) necessary to achieve $\tau_R \approx 0.1$ µsec.
Custom Dimensions and Polishing (Need for low-roughness surfaces for flow cells)	Precision Polishing (Ra < 1 nm for SCD)	Our SCD plates are available in custom dimensions and thicknesses (0.1 µm - 500 µm) with superior surface finish (Ra < 1 nm), crucial for minimizing scattering losses and ensuring stable attachment chemistry in microfluidic flow cells.

Engineering Support

6CCVD’s in-house PhD team provides expert consultation on material selection, doping strategies, and surface preparation protocols necessary to replicate or extend this single-molecule EPR research. We ensure that the diamond material meets the stringent quantum specifications required for advanced spin sensing applications.

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

Tech Support

Original Source

DOI: https://doi.org/10.1016/j.bpj.2016.03.022

References

2007 - A survey of single-molecule techniques in chemical biology [Crossref]
2003 - Stretching DNA and RNA to probe their interactions with proteins [Crossref]
2004 - Single-molecule spectroscopic methods [Crossref]
2006 - Single-molecule fluorescence studies of protein folding and conformational dynamics [Crossref]
2004 - An overview of the biophysical applications of atomic force microscopy [Crossref]
2004 - Single-molecule manipulation of nucleic acids [Crossref]
2004 - Mechanical processes in biochemistry [Crossref]
2004 - Structural dynamics and processing of nucleic acids revealed by single-molecule spectroscopy [Crossref]
2005 - Single-molecule RNA science [Crossref]
2008 - Single-molecule biophysics: at the interface of biology, physics and chemistry [Crossref]