In situ electron paramagnetic resonance spectroscopy using single nanodiamond sensors

At a Glance

Metadata	Details
Publication Date	2023-10-07
Journal	Nature Communications
Authors	Zhuoyang Qin, Zhecheng Wang, Fei Kong, Jia Su, Zhehua Huang
Institutions	University of Science and Technology of China
Citations	20
Analysis	Full AI Review Included

Executive Summary

This research presents a breakthrough methodology for performing Electron Paramagnetic Resonance (EPR) spectroscopy using single, randomly tumbling nanodiamond (ND) sensors, enabling robust in situ measurements.

Orientation Robustness: The core innovation is a generalized zero-field EPR technique utilizing Amplitude Modulation (AM) on the microwave (MW) control field, making the resonance condition independent of the ND sensor’s orientation.
Mechanism: The AM generates equidistant Floquet states, where the energy splitting (and thus the resonance frequency) is determined by the orientation-independent modulation frequency ($f$), rather than the anisotropic magnetic field response of the NV center.
In Situ Demonstration: Successfully acquired the zero-field EPR spectrum of vanadyl ions (VO²⁺) in an aqueous glycerol solution using embedded, tumbling single ND sensors.
Sensor Technology: Utilizes Nitrogen-Vacancy (NV) centers in ~40 nm nanodiamonds, offering single-spin sensitivity under ambient conditions (293 K).
Biological Relevance: This orientation-robust scheme eliminates a major hurdle for ND-based EPR, paving the way for nanoscale, in vivo EPR studies within complex biological environments, such as single cells.
Spectral Resolution: The use of a high-viscosity glycerol/water mixture (9:1) was critical to slow the rotational diffusion rate of the vanadyl ions (R_rot ~ 2 MHz), maintaining a manageable spectral linewidth (estimated $\le$66 MHz).

Technical Specifications

Parameter	Value	Unit	Context
Sensor Type	NV Centers in Nanodiamond	N/A	Single-spin sensitivity sensor.
Nanodiamond Size	~40	nm	Carboxylated Red Fluorescent NDs.
NV Center Density	12-14	N/A	Average NV centers per ND particle.
NV Zero-Field Splitting (D)	2.87	GHz	Intrinsic energy gap of the NV sensor.
Target Ion	Vanadyl Ion ([VO(H₂O)₅]²⁺)	N/A	Paramagnetic target molecule (S=1/2, I=7/2).
Target Concentration	25	mM	Concentration of vanadyl sulfate in solution.
Fitted Hyperfine Constant (A_perp)	195 ± 2	MHz	Hyperfine interaction perpendicular to the V=O axis.
Fitted Hyperfine Constant (A_parallel)	579 ± 8	MHz	Hyperfine interaction parallel to the V=O axis.
NV Decoherence Rate ($\Gamma_{2,NV}$)	~12	MHz	Estimated from the NV center resonance spectrum (fixed NDs).
Solution Viscosity ($\eta$)	0.3	Pa·s	9:1 Glycerol/water mixture (at 293 K).
Vanadyl Rotational Diffusion (R_rot)	~2	MHz	Calculated rate in the 9:1 glycerol/water mixture.
Estimated Linewidth (FWHM)	$\le$66	MHz	Total estimated linewidth for the vanadyl EPR signal.
Measurement Time (Fig. 3d)	7	days	Total time consumption for the tumbling ND experiment.
Duty Cycle	1:19	N/A	Ratio of MW pulse ON time to total time.

Key Methodologies

The experiment relies on a robust quantum control scheme combined with precise chemical surface engineering to stabilize the ND sensors in liquid environments.

Experimental Setup: A home-built confocal microscope system was used, integrating a 532 nm green laser for excitation and red fluorescence detection (PL readout). Microwave (MW) control was delivered via a coplanar waveguide driven by an arbitrary waveform generator and amplifier.
ND Surface Functionalization: Carboxylated NDs (~40 nm) were biotinylated using amine-PEG3-biotin, EDC, and MES buffer to prepare them for tethering.
Coverslip Preparation and Tethering: Coverslips were cleaned (KOH, Piranha solution) and amino-silanated (using APTES). Long-chain biotinylated PEG (20,000 Da) was bound to the surface to act as a soft “string” (length ~120 nm), restricting transnational motion while allowing rotational tumbling. Streptavidin was used as the linker to attach the biotinylated NDs to the PEG strings.
Vanadyl Solution Preparation: A 25 mM vanadyl sulfate (VO²⁺) solution was prepared in a 9:1 glycerol/water mixture. Solvents were deoxygenated (purging with N₂) and acidified (1 M sulfuric acid) to prevent ion oxidation and maintain stability.
Amplitude-Modulated (AM) Zero-Field EPR: The measurement sequence applied a continuous MW driving field (B₁ cos $\Omega$t) with an additional periodical amplitude modulation (B₁ cos $f$t cos $\Omega$t).
Spectrum Acquisition: The modulation frequency ($f$) was swept. When $f$ matched the target spin splitting ($\omega$), cross-relaxation occurred, resulting in a measurable reduction in the NV center’s photoluminescence (PL) rate, yielding the zero-field EPR spectrum.

Commercial Applications

The development of orientation-robust, ambient, nanoscale EPR sensing opens significant opportunities in several high-tech sectors.

In Vivo and Single-Cell Sensing:
- Monitoring redox reactions and molecular dynamics (e.g., vanadyl ion metabolism) directly inside living cells without requiring fixed orientation or cryogenic conditions.
- Nanoscale thermometry and relaxometry in complex biological environments.
Drug Discovery and Screening:
- High-throughput screening of drug candidates by monitoring their interaction with paramagnetic metal centers or radicals in biological molecules (e.g., proteins, membranes).
Materials Science and Polymer Dynamics:
- Studying molecular motion and spin dynamics at interfaces or within soft materials (e.g., polymers, lipid bilayers) where the sensor (ND) is free to tumble.
Quantum Sensing Technology:
- Advancing the utility of NV centers as robust quantum sensors for magnetic fields and spin noise under ambient, liquid-phase conditions, overcoming limitations imposed by traditional anisotropic response.
Chemical Catalysis:
- In situ analysis of structure-reactivity relationships in heterogeneous catalysis by monitoring paramagnetic intermediates on catalyst surfaces.

Tech Support

Original Source

DOI: https://doi.org/10.1038/s41467-023-41903-5